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A. we treated a panel of breast cancer cells MDA-MB-468, SUM190, KPL-4, and MDA-IBC-3 with different concentrations of selumetinib and assessed cell viability by WST-1 assay 72 hours later. We observed that in MDA-MB-468, SUM190, KPL-4, and MDA-IBC-3 cells, the 50% inhibitory concentration (IC50) was above 20 µM. B. MDA-MB-231 and SUM149 cells were treated with selumetinib for 72 hours, and FACS analysis was performed to determine cell cycle distribution using propidium iodide.

Background: Triple-negative breast cancer (TNBC) has high rates of relapse and metastasis. It has a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, plays a critical role in promoting CSC maintenance, cell proliferation, and malignant transformation. Here, we assessed the role of MELK in TNBC aggressiveness. Methods: The clinical relevance of MELK in TNBC was analyzed using the World IBC Consortium dataset (n = 314). The effects of MELK knockdown (siRNA), knockout (CRISPR-Cas9), and MELK inhibition with a novel selective small molecule inhibitor MELK-In-7 on migration and invasion were determined using the migration and invasion assays, respectively. MELK’s role in regulating CSC phenotypes was assessed using the mammosphere assay and flow cytometry. The impact of MELK knockdown on epithelial-to-mesenchymal transition (EMT) was determined using Western blotting. MELK’s protumorigenic role was determined in vitro using soft agar assay and in vivo using TNBC xenograft mouse models. Results: MELK mRNA expression level was higher in TNBC tumors than in tumors expressing hormone receptor, HER2, or both (P < 0.0001). In univariate analysis, breast cancer patients with high-MELK-expressing tumors had worse overall survival (OS, P < 0.001) and distant metastasis-free survival (DMFS, P < 0.01) than patients with low-MELK-expressing tumors, suggesting a prognostic impact of MELK in breast cancer. MELK knockdown using siRNA or MELK inhibition using MELK-In-7 significantly reduced migration, invasion, mammosphere formation, CD24-/CD44+ subpopulations, and ALDH activity, and reversed EMT in TNBC cells. This result suggests a role for MELK in regulating CSCs and EMT, which play crucial roles in tumor invasion and metastasis. Nude mice injected with MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and significantly improved OS compared with the mice injected with parental MDA-MB-231 cells (P < 0.05). Further, MELK-selective inhibitor (MELK-In-7) suppressed the growth of 4T1 mouse TNBC tumors in a dose-dependent manner in syngeneic BALB/c mice (P < 0.001). These results indicate an essential role for MELK in promoting TNBC tumor growth and metastasis. To uncover the underlying molecular mechanism of MELK’s regulation of TNBC metastasis, we performed microarray analysis and identified SERPINE1, a serine protease inhibitor known to regulate metastasis, as a downstream target of MELK. We validated that mRNA levels of SERPINE1 were reduced in MELK-knockout MDA-MB-231 cells and MELK-In-7-treated MDA-MB-231 cells. Moreover, SERPINE1 knockdown using siRNA reduced migration and invasion of TNBC cells. These results suggest that MELK regulates TNBC cell motility through SERPINE1. Conclusion: This work confirms MELK as a driver of aggressiveness and metastasis in TNBC. The mechanisms underlying MELK’s regulation of TNBC metastasis via SERPINE1 require further studies. Citation Format: Xuemei Xie, Gaurav B. Chauhan, Ramakrishna Edupuganti, Takahiro Kogawa, Jihyun Park, Moises T Tacam, Fnu Vidhu, Diane D. Liu, Juliana M. Taliaferro, Mary Kathryn Pitner, Yu Shen, Naoto T. Ueno, Savitri Krishnamurthy, Gabriel N. Hortobagyi, Debu Tripathy, Steven J. Van Laere, Geoffrey Bartholomeusz, Kevin Dalby, Chandra Bartholomeusz. Maternal embryonic leucine zipper kinase is associated with metastasis 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 P5-08-14.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer characterized by metastasis, drug resistance and high rates of recurrence. With a lack or targeted therapies, TNBC is challenging to treat and carries a poor prognosis. Patients with TNBC tumors expressing high levels of ERK2 have a poorer prognosis than those with low ERK2-expressing tumors. The MAPK pathway is often found to be highly activated in TNBC, however the precise functions of the ERK isoforms (ERK1 and ERK2) in cancer progression have not been well defined. We hypothesized that ERK2, but not ERK1, promotes the cancer stem cell (CSC) phenotype and metastasis in TNBC. Stable knockdown clones of the ERK1 and ERK2 isoforms were generated in SUM149 and BT549 TNBC cells using shRNA lentiviral vectors. ERK2 knockdown significantly inhibited anchorage-independent colony formation and mammosphere formation, indicating compromised self-renewal capacity. This effect correlated with a reduction in migration and invasion. SCID-beige mice injected via the tail vein with ERK clones were employed to determine metastatic potential. SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice or mice injected with SUM149 shERK1 cells. The Affymetrix HGU133plus2 microarray platform was employed to identify gene expression changes in ERK isoform knockdown clones. Comparison of gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown revealed differential and in some cases opposite effects on mRNA expression levels. Those changes associated with ERK2 knockdown predominantly altered regulation of CSCs and metastasis. Our findings indicate that ERK2 promotes metastasis and the CSC phenotype in TNBC.

There is an unmet need in triple-negative breast cancer (TNBC) patients for targeted therapies. Maternal embryonic leucine zipper kinase (MELK) is a promising target for inhibition based on the abundance of correlative and functional data supporting its role in various cancer types. Areas covered: This review endeavors to outline the role of MELK in cancer. Studies covering a range of biological functions including proliferation, apoptosis, cancer stem cell phenotypes, epithelial-to-mesenchymal transition, metastasis, and therapy resistance are discussed here in order to understand the potential of MELK as a clinically significant target for TNBC patients. Expert opinion: Targeting MELK may offer a novel therapeutic opportunity in TNBC and other cancers. Despite the abundance of correlative data, there is still much we do not know. There are a lack of potent, specific inhibitors against MELK, as well as an insufficient understanding of MELK's downstream substrates. Addressing these issues is the first step toward identifying a patient population that could benefit from MELK inhibition in combination with other therapies.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19–6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial–mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem cell–like characteristics and a CD44+CD24−/low expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44+CD24−/low fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent. Mol Cancer Ther; 14(12); 2773–81. ©2015 AACR.

Background: Triple-negative breast cancer (TNBC), which affects over 170 000 women worldwide every year, is considered the most arduous to treat subtype of breast cancer. With no targeted therapy, high rates of drug resistance and rapid metastasis, TNBC carries a poor prognosis. The MEK-ERK-MAPK signaling cascade is known to play a role in numerous cancers. Despite the lack of activating Ras/MAPK mutations in breast cancer, transcriptional signatures of this pathway are prevalent in TNBC. Our previous work showed that TNBC patients with tumors overexpressing ERK2 had a lower overall survival rate than did patients with low-ERK2-expressing tumors. MEK inhibitors selumetinib (AZD6244) and pimasertib (AS703026) are active in preclinical models, but not as single agents in the clinic. Using a synthetic lethal siRNA screen, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers. It is associated with cell immortalization, transformation, and chemoresistance. Patients with TNBC tumors expressing high levels of Mcl-1 have lower overall survival and distant-metastasis-free survival rates. We hypothesized that Mcl-1 promotes MEK inhibitor resistance in TNBC. Methods/Results: To model MEK inhibitor resistance, we established selumetinib- and pimasertib-resistant clones of SUM-149 and MDA-MB-231 TNBC cells by continuous exposure to increasing concentrations of inhibitors over a six month period. We confirmed the onset of MEK resistance by demonstrating that resistant cells, in comparison to the parental cells, exhibited no change in cell proliferation upon treatment with the MEK inhibitors. Resistant cells also displayed more effective cell migration and mammosphere formation than parental cells, suggesting a higher fraction of tumor-initiating cells. We found Mcl-1 to be highly expressed in 83% (15 of 18) of TNBC cell lines but only 30% (3 of 10) of other breast cancer cell lines. Resistant cells had higher levels of Mcl-1 than did parental cells. To determine whether Mcl-1 is required for MEK sensitivity, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. The Mcl-1 inhibitor restored MEK sensitivity in both resistant cell lines. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. Similar studies were done using an siRNA against Mcl-1. Conclusion: Our data demonstrate that Mcl-1 may promote TNBC resistance to MEK inhibitors and that Mcl-1 is a promising target for combination therapy. We will continue to explore the mechanisms of MEK inhibitor resistance by screening for additional genes/pathways involved. Our long-term goal is to design rational combination approaches to counteract the emergence of resistance by using novel molecularly targeted therapeutics. Citation Format: Gagliardi M, Chauhan G, Pitner MK, Iles L, Qi Y, Pusztai L, Tripathy D, Bartholomeusz G, Bartholomeusz C. Overcoming MEK inhibitor resistance in triple-negative breast cancer by targeting myeloid cell leukemia-1 (MCL1), an anti-apoptotic protein [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-06.

// Venkata Lokesh Battula 1,2 , Khoa Nguyen 1 , Jeff Sun 1 , Mary Kathryn Pitner 2 , Bin Yuan 1 , Chandra Bartholomeusz 2 , Numsen Hail 1 and Michael Andreeff 1 1 Department of Leukemia, Section of Molecular Hematology and Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Breast Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Venkata Lokesh Battula, email: // Michael Andreeff, email: // Keywords : breast cancer, cancer stem cells, GD2, GD3 synthase, NFκB Received : October 13, 2016 Accepted : March 01, 2017 Published : March 16, 2017 Abstract We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2 + and GD2 - cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2 + cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2 + cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro . Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function,and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

Currently, no targeted drug is available for triple-negative breast cancer (TNBC), an aggressive breast cancer that does not express estrogen receptor, progesterone receptor, or HER2. TNBC has high mitotic activity, and, because Aurora A and B mitotic kinases drive cell division and are overexpressed in tumors with a high mitotic index, we hypothesized that inhibiting Aurora A and B produces a significant antitumor effect in TNBC. We tested this hypothesis by determining the antitumor effects of KW-2450, a multikinase inhibitor of both Aurora A and B kinases. We observed significant inhibitory activities of KW-2450 on cell viability, apoptosis, colony formation in agar, and mammosphere formation in TNBC cells. The growth of TNBC xenografts was significantly inhibited with KW-2450. In cell-cycle analysis, KW-2450 induced tetraploid accumulation followed by apoptosis or surviving octaploid (8N) cells, depending on dose. These phenotypes resembled those of Aurora B knockdown and complete pharmaceutical inhibition of Aurora A. We demonstrated that 8N cells resulting from KW-2450 treatment depended on the activation of mitogen-activated protein kinase kinase (MEK) for their survival. When treated with the MEK inhibitor selumetinib combined with KW-2450, compared with KW-2450 alone, the 8N cell population was significantly reduced and apoptosis was increased. Indeed, this combination showed synergistic antitumor effect in SUM149 TNBC xenografts. Collectively, Aurora A and B inhibition had a significant antitumor effect against TNBC, and this antitumor effect was maximized by the combination of selumetinib with Aurora A and B inhibition. Mol Cancer Ther; 14(12); 2687–99. ©2015 AACR.

Background The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery. Methods To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks. Results Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis. Conclusion Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.

Centrosome amplification (CA) is a contributor to carcinogenesis, generating aneuploidy, and chromosome instability. Previous work shows that breast adenocarcinomas have a higher frequency of centrosome defects compared to normal breast tissues. Abnormal centrosome phenotypes are found in pre-malignant lesions, suggesting an early role in breast carcinogenesis. However, the role of CA in breast cancers remains elusive. Identification of pathways and regulatory molecules involved in the generation of CA is essential to understanding its role in breast tumorigenesis. We established a breast cancer model of CA using Her2-positive cells. Our goal was to identify centrosome cycle molecules that are deregulated by aberrant Her2 signaling and the mechanisms driving CA. Our results show some Her2+ breast cancer cell lines harbor both CA and binucleation. Abolishing the expression of Cdk4 abrogated both CA and binucleation in these cells. We also found the source of binucleation in these cells to be defective cytokinesis that is normalized by downregulation of Cdk4. Protein levels of Nek2 diminish upon Cdk4 knockdown and vice versa, suggesting a molecular connection between Cdk4 and Nek2. Knockdown of Nek2 reduces CA and binucleation in this model while its overexpression further enhances centrosome amplification. We conclude that CA is modulated through Cdk4 and Nek2 signaling and that binucleation is a likely source of CA in Her2+ breast cancer cells.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen receptor, progesterone receptor, and HER2 overexpression. Patients with TNBC have a generally poor prognosis due to metastasis, high rates of recurrence, and lack of FDA-approved targeted therapies. We previously showed using functional proteomics that patients with high-ERK2-expressing TNBC tumors had a higher risk of death than those with low-ERK2-expressing tumors. Moreover, ERK2 but not ERK1 plays an important role in epithelial-mesenchymal transition (EMT) and is required for acquisition of stem cell-like characteristics. Compared to other breast cancer subtypes, TNBC has a higher proportion of cancer stem cells (CSCs) and is linked to EMT, two critical features associated with breast cancer progression, metastasis, and recurrence in patients. The MAPK signaling pathway is activated in TNBC, but the roles of ERK isoforms in tumor progression and metastasis are not well defined. We hypothesized that ERK2 but not ERK1 promotes EMT, the CSC phenotype, and metastasis in TNBC. Methods and Results: Knockdown of ERK2 in SUM149 and BT549 TNBC cells significantly inhibited anchorage-independent colony formation (p Conclusions and Future Directions: Our findings support our hypothesis, indicating that ERK2 promotes EMT and the CSC phenotype through EGR1 and mediates metastasis in TNBC. Future studies will determine ERK activity and pathway engagement using a novel peptide sensor based on the Sox fluorophore. We will pursue a therapeutic approach using siRNA against ERK2 incorporated in a DOTAP:cholesterol liposome. Citation Format: Mary Kathryn Pitner, Hitomi Saso, Richard Larson, Rachel M. Sammons, Huiqin Chen, Caimiao Wei, Gaurav Chauhan, Kimie Kondo, Naoto T. Ueno, Kevin Dalby, Bisrat G. Debeb, Chandra Bartholomeusz. Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis 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 1624.

Purpose: Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the two most aggressive types of breast cancer whose molecular mechanisms remain unclear, representing a challenge for the development of effective targeted therapies. Single agent targeted therapies are of limited effectiveness in these types of breast cancer. Therefore, we aim to identify new combination therapeutic candidates for these aggressive diseases by comprehensive genomic screening. Experimental Design: We screened kinome siRNA libraries with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor [pimasertib], and genome-wide functional mRNA expression with the histone deacetylase (HDAC) type I inhibitor [entinostat] in TNBC and IBC cell lines. We evaluated the relationship between targets of interest and breast cancer patient survival using the IBC consortium database composed of breast cancer patient samples with clinical follow up. After identifying the targets, we assessed the combinational synergistic effect and its mechanism via cytotoxicity assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, western blotting, and mammary fat pad xenograft mouse models. Results: We identified that knock-down of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, enhanced the anti-proliferative effect of pimasertib. We observed that entinostat induced the expression of Noxa, a pro-apoptotic BH3-only member of the Bcl-2 family that is known to bind and degrade Mcl-1. Interestingly, in a breast cancer patient cohort (N = 389), high Mcl-1/low Noxa co-expression was associated with poorer survival outcomes than low Mcl-1/high Noxa co-expression (P = 0.0038). We found that combination with pimasertib and entinostat enhanced the inhibition of tumor cell proliferation (P < 0.001) compared with entinostat or pimasertib alone. We also observed significant in vivo tumor growth inhibition in both IBC (SUM190, P < 0.0001) and TNBC (SUM149, P < 0.05) xenograft models. Specifically, TNBC and IBC cell lines that overexpressed Noxa after treatment with entinostat were observed to be selectively sensitive to combination treatment with pimasertib. The synergistic antitumor activity of the entinostat- pimasertib combination was due to increased expression of Noxa, which induced the degradation of Mcl-1, resulting in the induction of mitochondrial cell death. Conclusion: Our data provide evidence that entinostat has enhanced antitumor effect in combination with pimasertib, resulting in the induction of apoptosis by Noxa-mediated Mcl-1 degradation. These findings provide a novel preclinical rationale for developing a clinical trial based on combinatorial HDAC and MEK inhibition therapy for TNBC and IBC with high Mcl-1 expression. Citation Format: Torres-Adorno AM, Lee JJ, Kogawa T, Bartholomeusz C, Pitner MK, Ordentlich P, Lim B, Tripathy D, Ueno NT. The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation. [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 P5-04-02.