Bruinsma, Elizabeth S.

Abstract Background: The tamoxifen (TAM) metabolite, ENDX, demonstrated promising antitumor activity in endocrine resistant breast cancer (BC) in both phase I and phase II settings. Furthermore, ENDX resulted in superior in vivo antitumor activity compared to TAM and letrozole in aromatase-expressing aromatase inhibitor-sensitive and resistant MCF7AC1 models. Recently, we identified protein kinase C beta 1 (PKCβ1), which regulates cell proliferation and tumorigenic transformation, as a novel target of ENDX. ENDX-bound PKCβ1 at concentrations achieved in phase I/II ENDX studies (100-300 nM). In contrast, TAM binding to PKCβ1 occurred at concentrations 7-10 folds higher (2 μM) than achievable with TAM 20 mg/day dosing. However, the clinical relevance of targeting PKCβ1 kinase activity is unclear, since drugs that target PKCβ1 (enzastaurin) have been ineffective in BC and other solid tumors. Therefore, we sought to understand how ENDX altered PKCβ1 and to further compare and contrast ENDXs effects to that of PKCβ1 kinase inhibition in ERα+ BC. Methods: The effects of PKCβ1 silencing and ENDX treatment on gene expression was analyzed by RNAseq in MCF7AC1 cells. The impact of PKCβ1-silencing on cell cycle was evaluated by flow cytometry. Protein expression of cell cycle regulators in PKCβ1 and ENDX-treated MCF7AC1 and T47D cells were compared to TAM and enzastaurin in vitro and to letrozole, TAM or control in vivo. The effects of PKCβ1 and drugs on growth were analyzed by cell proliferation assays. PRKCB gene amplification was assessed in primary tumors using TCGA data and in metastatic tumors using whole-exome sequencing data from patients enrolled in the PROMISE study (NCT 03281902). Results: RNAseq analysis revealed E2F targets and G2M checkpoints as the top hallmark genesets significantly downregulated in both PKCβ1-silenced and ENDX-treated MCF7AC1 cells. Flow cytometry demonstrated that PKCβ1 silencing increased G1 and reduced S phases of the cell cycle. Western blot analyses of PKCβ1-silenced MCF7AC1 and T47D cells displayed reduced protein levels of the cell cycle regulators Cyclin D1, Retinoblastoma (Rb), phospho-RbS807/811, CDK4, Chk1 and E2F1 that regulate G1/S transition. While short term ENDX (48 hours) treatment did not alter PKCβ1 levels, prolonged in vitro ENDX treatment profoundly reduced PKCβ1 protein levels and the aforementioned cell cycle regulators, faithfully replicating PKCβ1 silencing effects. In contrast, enzastaurin had no impact on proliferation or cell cycle proteins in either model. Consistent with this finding, ENDX, but not TAM or letrozole, reduced protein levels of ERα and cell cycle regulators in vivo. Overexpression of PKCβ1 induced TAM, but not ENDX, resistance and had little impact on responsiveness to enzastaurin. While PRKCB gene amplification was uncommon in newly diagnosed ERα+/HER2- BC (5%, TCGA), PRKCB was amplified in 40% of metastatic ERα+/HER2- BC (PROMISE study). Conclusion: We have confirmed the relevance of a new ENDX target, PKCβ1, in ERα+/HER2- BC. While targeting PKCβ1 kinase activity elicited no anticancer effects in ERα+ cells, PKCβ1 downregulation, either by siRNA or ENDX, resulted in profound ERα turnover, reduced protein levels of essential cell cycle mediators and profoundly inhibited cell proliferation. Furthermore, PKCβ1 protein expression is associated with TAM, but not ENDX, resistance, a finding whose clinical relevance is further magnified by identification of PRKCB amplification in metastatic ERα+ BC, confirming its potential importance in progression. Efforts are currently underway to elucidate the mechanistic basis for ENDX-induced PKCβ1 and ERα degradation and the contribution of these effects to the superior antitumor activity of ENDX in ERα+ BC. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Vera J Suman, Elizabeth S Bruinsma, Ciara C O'Sullivan, Liewei Wang, Richard Weinshilboum, James N Ingle, John R Hawse, Matthew P Goetz. Antitumor activity of Z-endoxifen (ENDX) is mediated via PKCβ1-dependent ERα loss and cell cycle arrest in ERα-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-04.

Abstract Background: Epigenetic based therapies, such as histone deacetylase inhibitors, have proven successful in many cancer types including lymphomas and myelomas with ongoing phase III clinical trials in NSCLC. Epigenetic modulation in triple negative breast cancer (TNBC) has been increasingly explored in the past decade. A major issue preventing the uptake of these drugs in the clinic is the relatively high doses necessary to achieve therapeutic benefit. It is therefore of interest to identify combinatorial approaches that result in drug de-escalation and enhanced therapeutic benefit. We and others have demonstrated that estrogen receptor beta (ERβ) is expressed in approximately 20% of TNBC patients. ERβ targeted therapies, including estradiol and multiple ERβ selective agonists, result in suppression of cell proliferation, invasion, and migration in vitro and in vivo (Reese, PNAS 2018). For these reasons, we sought to determine if ERβ targeted therapies would synergize with epigenetic based therapies for the treatment of TNBC. Methods: Using multiple TNBC cell lines representative of ERβ+ and ERβ- disease, as well as a model of acquired resistance to ERβ targeted therapies, we performed an epigenetic drug screen assessing 145 compounds alone and in combination with estradiol treatment. The top hit from this screen was extensively profiled across multiple cell lines in the presence and absence of ERβ targeting agents and was compared to the efficacy observed in ERα+ models of breast cancer using cell proliferation assays, RT-PCR, and western blotting. Results: The most significant compound to elicit differential and therapeutically beneficial effects in ERβ+ TNBC was CUDC-101, a multitarget pan-HDACi, EGFRi, and HER2i that was developed based on the structures of Vorinostat, Erlotinib, and Lapatinib. CUDC-101 potently inhibits the growth of TNBC cells and synergizes with E2 stimulation in ERβ+ MDA-MB-231 TNBC cells, but not in ERβ- cells. CUDC-101 was also effective in ERβ-resistant MDA-MB-231 cells and restored the growth inhibitory effects of estrogen in this model. Treatment with Vorinostat (SAHA) alone elicited a similar, though far less potent, synergistic effect in the MDA-MB-231-ERβ+ cell line, suggesting that histone deacetylase inhibition is a critical, but not completely sufficient, component of the synergistic phenotype. CUDC-101, while effective at reducing the viability of MDA-MB-468-ERβ+ cell line, had no synergistic effect with E2 in this model suggesting differences in response between TNBC sub-types. Additionally, CUDC-101 was found to be ineffective in models of ERα+ disease (MCF7 and MCF7-ERβ) in both the presence and absence of E2 treatment. Conclusions: Therapeutic options remain extremely limited for TNBC patients and the development of novel treatment strategies for these patients continue to represent a major unmet clinical need. Here, we present evidence that CUDC-101 elicits potent anti-cancer effects in multiple models of TNBC and synergizes with ERβ-targeted therapies in some models. Our findings further strengthen the body of evidence suggesting ERβ as a valid therapeutic target and important biomarker in TNBC and further implicate HDACi/EGFRi as relevant therapeutic strategies within this patient population. Citation Format: Michael Justin Emch, David Benjamin, Elizabeth Bruinsma, Molly Nelson-Holte, Matthew Goetz, John Hawse. CUDC-101 enhances estrogen-mediated anticancer effects in ERβ+ TNBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-10.

Abstract Background: The first in-human phase-I clinical trial of the tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in both aromatase inhibitor (AI) and tamoxifen resistant ER+ breast cancer patients (Goetz M.P., et al; JCO, 2017). By utilizing a letrozole-sensitive MCF7 aromatase expressing (MCF7AC1) cell line, and its corresponding letrozole-resistant (MCF7LR) cell line, we have shown superior antitumor and antiestrogenic activity of Z-endoxifen over tamoxifen in the AI-sensitive and resistant settings in vivo. This study also revealed that Z-endoxifen may target protein kinases in addition to estrogen receptor alpha. Follow-up studies demonstrated that Z-endoxifen, but not tamoxifen, binds to protein kinase C beta 1 (PKCβ1), a member of the serine/threonine-specific protein kinase family that regulates signaling pathways involved in cell proliferation and tumorigenic transformation (Guo C., et al; SABCS, 2017). We have further shown that PKCβ1 silencing, similar to Z-endoxifen, potently inhibits ER+ cell proliferation and additionally causes ERα turnover. Here, we report a potential mechanism by which PKCβ1 may mediate its anti-proliferative effects in ER+ breast cancer cells. Methods: The effects of siRNA-mediated PKCβ1-silencing on gene transcription were evaluated by RNAseq in MCF7AC1 cells. RNAseq results were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in PKCβ1-silenced MCF7AC1 cells as well as in ER+ T47D cells. The effects of Z-endoxifen on the mRNA expression of genes altered by PKCβ1-silencing in MCF7AC1 cells were evaluated by qRT-PCR analysis. PKCβ1 silencing effects on protein expression in MCF7AC1 cells were evaluated by immunoblot analysis. Results: RNAseq analysis of PKCβ1-silenced MCF7AC1 cells compared to non-silenced cells revealed NF-κB signaling pathway as the top biological pathway significantly altered by PKCβ1 silencing (p=5.93e-08). Enhanced mRNA expression of multiple NF-κB downstream target genes including CXCL10, RELB, MMP1 and IL6 were observed following PKCβ1 silencing. qRT-PCR analyses validated increased mRNA expression of these genes in PKCβ1-silenced MCF7AC1 and T47D cells. Notably, Z-endoxifen treatment in MCF7AC1 cells mirrored PKCβ1 silencing effects, resulting in enhanced mRNA expression of the aforementioned genes. Evaluation of the protein expression of RelA and RelB, the canonical and noncanonical NF-κB transcription factors respectively, in PKCβ1-silenced MCF7AC1 cells revealed significant upregulation of RelA (p=0.0203) but not RelB (p=0.1360) expression. ERα silencing in MCF7AC1 cells did not affect RelA protein expression. Consistent with prior reports, treatment of MCF7 cells with TNFα inhibited cell proliferation. Finally, targeted siRNA-mediated silencing of RelA in PKCβ1-silenced MCF7AC1 cells significantly attenuated PKCβ1-mediated growth inhibition (p=0.0002). Conclusion: Our findings demonstrate that both Z-endoxifen treatment and PKCβ1-silencing commonly activates a NF-κB-mediated gene signature in ER+ breast cancer cells. Notably, PKCβ1-silencing induces the protein expression of the NF-κB transcription factor RelA in an ERα-independent manner, which appears to contribute to PKCβ1-mediated growth inhibition. Current studies are ongoing to evaluate the impact of RelA silencing on Z-endoxifen mediated growth inhibition. This information will foster our understanding of the estrogenic and non-estrogenic mechanisms by which Z-endoxifen mediates its antitumor activity in ER+ breast cancer. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Elizabeth S Bruinsma, John R Hawse, Matthew P Goetz. Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-06.

AbstractAimTieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood.MethodsWe have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed.ResultsLoss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1H‐NMR spectra revealed no significant metabolic difference between wild‐type and KO muscles. However, 31P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice.ConclusionOur findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.

Significance Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and patients exhibit high rates of recurrence and mortality in part due to lack of treatment options beyond standard-of-care chemotherapy regimens. In the subset of TNBCs that express estrogen receptor beta (ERβ), ligand-mediated activation of ERβ elicits potent anticancer effects. We report here the elucidation of the ERβ cistrome and transcriptome in TNBC and identify a mechanism whereby ERβ induces cystatin gene expression resulting in inhibition of canonical TGFβ signaling and a blockade of metastatic phenotypes. These findings suggest that ERβ-targeted therapies represent a treatment option for the subset of women with ERβ-expressing TNBC.

TIEG knockout (KO) mice exhibit a female‐specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up‐regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG‐SW3 osteocyte cells using shRNA and CRISPR‐Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen‐regulated miRNAs in modulating SOST expression in bone.

Abstract Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells’ documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells.

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.