Citations

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple-negative breast cancer (TNBC) is an aggressive subtype representing 15-20% of all breast cancers. TNBC often ends with a poor clinical outcome due to high histological grade and recurrence rates with few treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance, and alternative treatment strategies for the chemoresistant disease remain a major unmet need. circRNAs are newly identified non-coding RNA molecules with covalently closed circular structures. Recently, an increasing number of studies have indicated that circRNAs play crucial roles in regulating tumor development and chemoresistance. However, the role of circRNAs in the process of TNBC chemotherapy resistance is not yet fully clear. Materials and methods: To study the molecular functions of circRNAs in TNBC chemoresistance, identify alternative biomarkers for chemotherapy-resistant disease, and elucidate novel therapeutic targets, doxorubicin-resistant (Doxo-R) and paclitaxel-resistant (Taxol-R) cell lines were generated using TNBC models. Results: Doxo-R and Taxol-R cells exhibited a 3- and 12-fold decrease in sensitivity to their respective chemotherapeutic agents. We identified a large spectrum of gene expression changes that were acquired in Doxo-R and Taxol-R cells. The expression of circRNAs was profiled using circRNA microarrays, and top hits were validated using RT-PCR. We identified 429 and 310 circRNAs that were differentially expressed in Doxo-R and Taxol-R resistant cells respectively compared to the parental chemosensitive cell line (|FC| ≥ 1.5; p value < 0.05). Of these, 66 were commonly differentially expressed between the two chemotherapy resistant models. Conclusions: These results revealed that Doxo-R and Taxol-R TNBC cell lines were successfully established and serve as good models for studying the mechanisms of chemotherapy resistance and the regulatory roles of circRNAs in the development of chemoresistance in TNBC. Increasing knowledge of the important functions of circRNAs underlying drug resistance will provide new opportunities for developing efficacious therapeutic strategies and prognostic/predictive biomarkers for TNBC. Citation Format: Xiyin Wang, Michael Emch, John Hawse. The role of circular RNAs in triple-negative breast cancer and chemotherapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-02.

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple negative breast cancer (TNBC) is an aggressive subtype representing 15-20% of all breast cancers. TNBC often ends with a poor clinical outcome due to high histological grade and recurrence rates with few treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance, and alternative treatment strategies for chemoresistant disease remain a major unmet need. ERβ, a second form of the estrogen receptor, is known to be expressed in approximately 20-30% of TNBC, and ligand-mediated activation of ERβ elicits potent anti-cancer effects in chemosensitive TNBC. However, the utility of ERβ in the chemoresistant setting remains unclear. Materials and methods: To study the mechanistic basis of chemoresistance in TNBC cells and identify alternative treatment approaches for chemotherapy-resistant disease, doxorubicin-resistant (Doxo-R) and paclitaxel-resistant (Taxol-R) cell lines were generated using MDA-MB-231 cells. A Doxycycline (dox) inducible expression system was used to overexpress ERβ in these cells. RNA sequencing and transcription factor (TF) activation profiling was performed to glean a comprehensive mechanistic insight into mechanisms of resistance and the functions of ERβ, and the efficacy of ERβ targeted therapies, were assessed. Results: Doxo-R and Taxol-R cells exhibited a 3- and 12-fold decrease in sensitivity to their respective chemotherapeutic agents. Doxo-R cells were more sensitive to paclitaxel while Taxol-R cells exhibited no changes in sensitivity to doxorubicin. Expression of ERβ had no impact on the IC50s for either chemotherapy drug. Taxol-R cells exhibited a significantly increased number of cells in the S phase. Proliferation assays revealed that ERβ targeting agents, including estradiol (E2) and the ERβ-specific agonist LY500307 (LY), led to substantial inhibition of cell proliferation, migration, and invasion when ERβ was expressed. We identified a large spectrum of genes expression changes that were acquired in Doxo-R and Taxol-R cells. These changes included 91 kinases genes in Doxo-R cells and 164 in Taxol-R cells. TF activation profiling indicated that eight and four TFs were activated more than 2-fold, including NRF1, WT1, and SATB1 in both Doxo-R and Taxol-R cells. Conclusions: Doxo-R and Taxol-R TNBC cell lines were successfully established and serve as a good model for studying the mechanisms of chemotherapy resistance and screening effective agents to reverse or treat chemoresistant disease. As in the sensitive setting, ERβ targeted therapies may represent an alternative treatment for TNBC patients with chemorefractory ERβ+ disease, a possibility that is currently being explored through a phase II clinical trial (NCT03941730). Citation Format: Xiyin Wang, Michael J. Emch, John R. Hawse. Functional characteristics of chemotherapy-resistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1784.

Abstract Ovarian cancer is the 7th most common cancer in women. It is the most fatal of all female reproductive cancers, due to the 5-year overall survival of 10-30% and over half of patients being diagnosed with late-stage disease. Despite initial response to front line platinum- and taxol-based chemotherapies, over 70% of patients develop recurrent and chemotherapy resistant disease and nearly all of these women die of their disease. Recently, the use of targeted therapies such as bevacizumab and PARP inhibitors have been shown to improve progression-free survival. However, these regimens have thus far failed to improve overall survival in patients without specific genetic signatures, highlighting the urgent need for alternative therapies. We have identified lestaurtinib as a potent inhibitor of many ovarian cancer cell lines, including platinum and PARP inhibitor resistant models, and patient derived organoid models. RNA-sequencing following lestaurtinib treatment identified JAK/STAT signaling as one of the most profoundly downregulated pathways. Additionally, it was recently reported via single cell RNA sequencing of patient tumors that downstream mediators of the JAK/STAT pathway are among the most highly expressed genes in ovarian tumor cells. Further, we have discovered that STAT1 and STAT3 are constitutively activated in chemotherapy and PARP inhibitor resistant cell lines as reflected by high levels of Tyr701/705 and Ser727 phosphorylation suggesting induction of this pathway may drive resistance. Selective siRNA-mediated knockdown of STAT1/3 resulted in significant growth inhibition confirming their importance in maintaining cell viability. Surprisingly, we have identified profound differences in the ability of various JAK/STAT inhibitors to suppress ovarian cancer cell growth. For example, ruxolitinib, the only JAK/STAT inhibitor currently in clinical trials for ovarian cancer, failed to inhibit the growth sensitive, platinum resistant or PARP inhibitor resistant cell lines. Our preliminary data indicate that JAK/STAT inhibitor efficacy in preventing ovarian cancer cell growth is related to their ability to block specific phosphorylation events on STAT1/3 proteins. We believe that precise and selective inhibition of STAT1 and STAT3 signaling represents a robust, durable, and novel approach for the treatment of highly aggressive and resistant forms of ovarian cancer. Further we plan to elucidate the molecular mechanisms by which STAT1 and STAT3 function to support ovarian cancer cell viability and growth, in order to substantially advance our understanding of JAK/STAT signaling in ovarian cancer and to identify the most effective ways to pharmacologically inhibit JAK/STAT signaling in ovarian cancer cells. Outcomes from this work will have both immediate and long-term impacts for our patients and will lay the foundation for future biomarker-driven clinical trials. Citation Format: Esther Rodman, Michael Emch, Elizabeth Bruinsma, Xiaonan Hou, John Weroha, John Hawse. Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1129.

Abstract Background:. Poor patient outcomes in triple negative breast cancer (TNBC) largely stem from a lack of understanding of therapeutic vulnerabilities and an insufficient armamentarium of effective drugs. The standard of care for most early stage TNBC patients continues to be (neo)adjuvant chemotherapy and radiation. Identifying additional therapeutic options for this subset of patients represents a major unmet need. We and others demonstrated that estrogen receptor beta (ERβ) is expressed in about 20% of TN tumors. Prior research has shown that ligand-mediated activation of ERβ decreases proliferation, invasion, and migration in TNBC cell lines. In vivo, ERβ suppresses the growth of cell line xenograft models and prevents the development of metastatic lesions in a ligand dependent manner. Mechanistically, we found that ERβ repurposes EZH2 to suppress oncogenic NFκB signaling and that pharmacologic inhibition of EZH2 diminishes ERβ function and its anti-cancer effects. This research has led to an ongoing clinical trial (NCT03941730) assessing the efficacy of estradiol in ERβ+ TNBC patients with chemorefractory disease. As with all therapies, patients will exhibit de novo and acquired resistance and therefore we sought to understand the mechanisms of resistance to ERβ targeted therapies. Methods:. Using multiple models of ERβ positive TNBC, we developed ERβ resistant cell lines through chronic exposure to estradiol and the ERβ specific agonist LY500307 over a period of 8 months. We employed RNA sequencing to characterize the transcriptomic changes which occurred following ERβ resistance. We profiled XIST expression across multiple publicly available datasets, including TCGA, Metabric, BEAUTY, and GTEx. XIST expression was also modulated using CRISPR/Cas9 to assess subsequent effects on TNBC cell biology, ERβ function, and response to ERβ targeted therapies. Results:. Our resistant cell line models of ERβ positive TNBC maintained ERβ expression, but were no longer growth inhibited by ERβ agonists. RNAseq revealed substantial differences comparing the transcriptome of resistant versus sensitive cell lines, of which the most increased transcript in the resistant setting was the lncRNA XIST. XIST is best known for its role in X-chromosome inactivation through recruitment and association with the PRC2 complex, and thus EZH2. However, little is known about its functions in breast cancer. We therefore assessed the expression levels of XIST in breast tumors and cell lines. XIST expression was highly variable in breast cancer, was found in a proportion of all breast cancer sub-types and did not correlate with ERβ status. However, XIST expression was up-regulated in ERβ expressing cell lines following long term estrogen treatment. No effects on XIST expression were identified in multiple ERα positive models following estrogen, SERM/SERD treatment, or estrogen deprivation. Furthermore, upregulation of XIST was not associated with resistance to Paclitaxel or Doxorubicin, suggesting XIST is not a part of a broad resistance phenotype. Strikingly, CRISPR mediated knockout of XIST in ERβ resistant cells completely re-sensitized cells to ERβ targeted therapies suggesting that XIST expression is a critical component of ERβ resistance. Conclusions:. ERβ represents a relevant therapeutic target that is being tested in clinical trials. Using multiple in vitro models, we provide evidence that XIST expression is sufficient to induce resistance to. ERβ targeted therapies in TNBC and may therefore represent a relevant biomarker for patient stratification. Further strategies to suppress XIST expression may elicit anti-cancer effects on their own and may resensitize a sub-set of ERβ resistant tumors to ERβ agonists. Citation Format: Michael J Emch, Kirsten GM Aspros, Elizabeth S Bruinsma, Krishna R Kalari, Calley J Jones, Brandon W Simone, Matthew P Goetz, John R Hawse. The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-09.

Abstract Despite the availability of drugs that target ERα-positive breast cancer, resistance commonly occurs, resulting in relapse, metastasis, and death. Tamoxifen remains the most commonly-prescribed endocrine therapy worldwide, and “tamoxifen resistance” has been extensively studied. However, little consideration has been given to the role of endoxifen, the most abundant active tamoxifen metabolite detected in patients, in driving resistance mechanisms. Endoxifen functions differently from the parent drug and other primary metabolites, including 4-hydroxy-tamoxifen (4HT). Many studies have shown that patients who extensively metabolize tamoxifen into endoxifen have superior outcomes relative to patients who do not, supporting a primary role for endoxifen in driving tamoxifen responses. Therefore, “tamoxifen resistance” may be better modeled by “endoxifen resistance” for some patients. Here, we report the development of novel endoxifen-resistant breast cancer cell lines and have extensively compared these models to 4HT and fulvestrant (ICI)-resistant models. Endoxifen-resistant cells were phenotypically and molecularly distinct from 4HT-resistant cells and more closely resembled ICI-resistant cells overall. Specifically, endoxifen resistance was associated with ERα and PR loss, estrogen insensitivity, unique gene signatures, and striking resistance to most FDA-approved second- and third-line therapies. Given these findings, and the importance of endoxifen in the efficacy of tamoxifen therapy, our data indicate that endoxifen-resistant models may be more clinically relevant than existing models and suggest that a better understanding of endoxifen resistance could substantially improve patient care. Implications: Here we report on the development and characterization of the first endoxifen-resistant models and demonstrate that endoxifen resistance may better model tamoxifen resistance in a subset of patients.

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.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale . Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.