Hope E. Burks

Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.

Laser Direct-Write Onto Live Tissues: A Novel Model for Studying Cancer Cell Migration

Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct‐write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333–2338, 2016.

Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1

Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a −3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.

Endocrine disruptors and the tumor microenvironment: A new paradigm in breast cancer biology

Breast cancer is one of the most frequently diagnosed malignancies in women and is characterized by predominantly estrogen dependent growth. Endocrine disruptors (EDCs) have estrogenic properties which have been shown to increase breast cancer risk. While the direct effects of EDCs on breast cancer cell biology and tumor progression have been well studied, the roles for EDCs on tumor microenvironment composition, signaling and structure are incompletely defined. Estrogen targeting of tumor stromal cells can drive paracrine signaling to breast cancer cells regulating tumorigenesis and progression. Additionally, estrogen and estrogen receptor signaling has been shown to alter breast architecture and extracellular matrix component synthesis. Unsurprisingly, EDCs have been shown to induce structural changes in the mammary gland as well as increased collagen fibers in the tissue stroma. Previous work demonstrates that human mesenchymal stem cells (hMSC) are essential components of the tumor microenvironment and are direct targets of both estrogens and EDCs. Furthermore, estrogen-stem cell cross talk has been implicated in breast cancer progression and results in increased tumor cell proliferation, angiogenesis and invasion. This review aims to dissect the possible relationship and mechanisms between EDCs, the tumor microenvironment, and breast cancer progression.

Dual Src Kinase/Pretubulin Inhibitor KX-01, Sensitizes ERα-negative Breast Cancers to Tamoxifen through ERα Reexpression

Unlike breast cancer that is positive for estrogen receptor-α (ERα), there are no targeted therapies for triple-negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to reexpression of ERα in TNBC. KX-01 is a peptide substrate–targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-twice daily) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in nude mice that was correlated with Src kinase inhibition. KX-01 also increased ERα mRNA and protein, as well as increased the ERα targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1), and c-myc (MYC) in MDA-MB-231 and MDA-MB-468, but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, β-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERα targets pS2, cyclin D1, and c-myc. Chromatin immunoprecipitation (ChIP) of the ERα promoter in KX-01–treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERα promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERα expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen. Implications: Src kinase/pretubulin inhibitor KX-01 restores functional ERα expression in ERα– breast tumors, a novel treatment strategy to treat triple-negative breast cancer. Mol Cancer Res; 15(11); 1491–502. ©2017 AACR.

Abstract 5079: Inhibition of HDAC2, but not HDAC1, abrogates triple negative breast cancer progression and metastasis

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with higher rates of metastasis and recurrence than other classified subtypes. The decreased survival rates seen in TNBC can be attributed in part to the lack of targeted therapeutics. HDAC inhibitors (HDACi) have emerged as a potential adjuvant therapy for patients with triple negative disease. Previously, our lab has identified Panobinostat, a pan-DAC inhibitor, as a successful therapy for the reversal of the metastatic phenotype in TNBC. However, this therapy lacks specificity and may have increased side effects as a result. In this study, we aim to dissect the necessary HDACs responsible for driving and maintaining the metastatic phenotype of TNBC, with the goal of informing specific HDAC targeted therapies. Here we show that Romidepsin, an HDAC 1 and 2 specific inhibitor, is capable of recapitulating aspects of Panobinostat treatment in TNBC both in vitro and in a patient derived xenograft model in vivo. To parse the necessity and role of HDAC1 and HDAC2 individually, we created knockdown cell lines and analyzed them for changes in gene expression changes and their respective associated biological processes, including proliferation, migration, mammosphere formation, tumor growth and metastasis. We found that HDAC2 knockdown was able to significantly repress migration and proliferation in TNBC cells and their associated gene expression signatures in vitro. Furthermore, HDAC2 knockdown resulted in decreased tumor growth and metastasis an in vivo mouse model. These results illustrate the efficacy of Romidepsin in inhibition of oncogenic processes and implicate HDAC2 as a potential target for therapeutic intervention in TNBC. Citation Format: Hope E. Burks, Steven Elliott, Van T. Hoang, Margarite D. Matossian, Bridgette Collins Burow, Matthew E. Burow. Inhibition of HDAC2, but not HDAC1, abrogates triple negative breast cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5079. doi:10.1158/1538-7445.AM2017-5079

Abstract 1596: Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Hope E. Burks, Margarite Matossian, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Lowry Curley, Muralidharan Anbalagan, Brian G. Rowan, Doug Chrisey, Jane E. Cavanaugh, Patrick T. Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer. [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 1596.

Abstract 684: Targeting an unconventional kinase-invasion axis in breast cancer metastasis

Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer. Citation Format: Margarite D. Matossian, Steven Elliott, Van T. Hoang, Hope E. Burks, Theresa B. Phamudy, Doug Chrisey, William J. Zuercher, David H. Drewry, Carrow Wells, Bridgette Collins-Burow, Matthew E. Burow. Targeting an unconventional kinase-invasion axis in breast cancer metastasis. [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 684.