Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat

INTRODUCTION: Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo. METHODS: TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology. RESULTS: Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype. CONCLUSIONS: This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types

microRNA regulation of mammalian target of rapamycin expression and activity controls estrogen receptor function and RAD001 sensitivity

Background: The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17 α -estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer. Methods and results: Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor- α positive (ER α + ) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ER α + breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ER α signalingbothinculturedcells in vitro and in xenografts in vivo in addition to repressed PgR expression and act ivity.FurthermoreweobservedthatmiR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ER α -signaling pathway. Conclusions: miRNA mediated alterations in mTOR and ER α signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation

Ponderomotive effects in multiphoton pair production

The Dirac-Heisenberg-Wigner formalism is employed to investigate electron-positron pair production in cylindrically symmetric but otherwise spatially inhomogeneous, oscillating electric fields. The oscillation frequencies are hereby tuned to obtain multiphoton pair production in the nonperturbative threshold regime. An effective mass as well as a trajectory-based semi-classical analysis are introduced in order to interpret the numerical results for the distribution functions as well as for the particle yields and spectra. The results, including the asymptotic particle spectra, display clear signatures of ponderomotive forces.Comment: 9 pages, 3 Tables, 3 Figure

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype

NEK Family Review and Correlations with Patient Survival Outcomes in Various Cancer Types

The Never in Mitosis Gene A (NIMA)–related kinases (NEKs) are a group of serine/threonine kinases that are involved in a wide array of cellular processes including cell cycle regulation, DNA damage repair response (DDR), apoptosis, and microtubule organization. Recent studies have identified the involvement of NEK family members in various diseases such as autoimmune disorders, malignancies, and developmental defects. Despite the existing literature exemplifying the importance of the NEK family of kinases, this family of protein kinases remains understudied. This report seeks to provide a foundation for investigating the role of different NEKs in malignancies. We do this by evaluating the 11 NEK family kinase gene expression associations with patients’ overall survival (OS) from various cancers using the Kaplan–Meier Online Tool (KMPlotter) to correlate the relationship between mRNA expression of NEK1-11 in various cancers and patient survival. Furthermore, we use the Catalog of Somatic Mutations in Cancer (COSMIC) database to identify NEK family mutations in cancers of different tissues. Overall, the data suggest that the NEK family has varying associations with patient survival in different cancers with tumor-suppressive and tumor-promoting effects being tissue-dependent

Elevated expression of long intergenic non-coding RNA HOTAIR in a basal-like variant of MCF-7 breast cancer cells

Epigenetic regulation of gene expression is critical to phenotypic maintenance and transition of human breast cancer cells. HOX antisense intergenic RNA (HOTAIR) is a long intergenic non-coding RNA that epigenetically represses gene expression via recruitment of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase. Elevated expression of HOTAIR promotes progression of breast cancer. In the current study we examined the expression and function of HOTAIR in MCF-7-TNR cells, a derivative of the luminal-like breast cancer cell line MCF-7 that acquired resistance to TNF-α-induced cell death. The expression of HOTAIR, markers of the luminal-like and basal-like subtypes, and growth were compared between MCF-7 and MCF-7-TNR cells. These variables were further assessed upon inhibition of HOTAIR, EZH2, p38 MAPK, and SRC kinase in MCF-7-TNR cells. When compared with MCF-7 cells, MCF-7-TNR cells exhibited an increase in the expression of HOTAIR, which correlated with characteristics of a luminal-like to basal-like transition as evidenced by dysregulated gene expression and accelerated growth. MCF-7-TNR cells exhibited reduced suppressive histone H3 lysine27 trimethylation on the HOTAIR promoter. Inhibition of HOTAIR and EZH2 attenuated the luminal-like to basal-like transition in terms of gene expression and growth in MCF-7-TNR cells. Inhibition of p38 and SRC diminished HOTAIR expression and the basal-like phenotype in MCF-7-TNR cells. HOTAIR was robustly expressed in the native basal-like breast cancer cells and inhibition of HOTAIR reduced the basal-like gene expression and growth. Our findings suggest HOTAIR-mediated regulation of gene expression and growth associated with the basal-like phenotype of breast cancer cells

ZEB2 regulates endocrine therapy sensitivity and metastasis in luminal a breast cancer cells through a non-canonical mechanism

PURPOSE: The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. METHODS: In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17β-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. RESULTS: Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17β-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. CONCLUSION: ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms

Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways

Triple negative breast cancer (TNBC) patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression

NEK5 activity regulates the mesenchymal and migratory phenotype in breast cancer cells

Purpose Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer. Methods Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis. Results NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively. Conclusion While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression