Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Tetraploidization increases sensitivity to Aurora B kinase inhibition

Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells.     

Aurora Kinases as Druggable Targets in Pediatric Leukemia: Heterogeneity in Target Modulation Activities and Cytotoxicity by Diverse Novel Therapeutic Agents

Leukemia is the most common pediatric malignancy, constituting more than 30% of all childhood cancers. Although cure rates have improved greatly, approximately one in five children relapse and poor survival rates post relapse remain a challenge. Given this, more effective and innovative therapeutic strategies are needed in order to improve prognosis. Aurora kinases, a family of serine/threonine kinases essential for the regulation of several mitotic processes, have been identified as potential targets for cancer therapeutics. Elevated expression of Aurora kinases has been demonstrated in several malignancies and is associated with aberrant mitotic activity, aneuploidy and alterations in chromosomal structure and genome instability. Based on this rationale, a number of small molecule inhibitors have been formulated and advanced to human studies in the recent past. A comparative analysis of these agents in cytotoxicity and target modulation analyses against a panel of leukemia cells provides novel insights into the unique mechanisms and codependent activity pathways involved in targeting Aurora kinases, constituting a distinctive preclinical experimental framework to identify appropriate agents and combinations in future clinical studies.     

Featured Article: Teriflunomide,an immunomodulatory drug,exerts anticancer activity in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC in vitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.     

Aurora kinases link chromosome segregation and cell division to cancer susceptibility

Aurora kinases play critical roles in chromosome segregation and cell division. They are implicated in the centrosome cycle, spindle assembly, chromosome condensation, microtubule-kinetochore attachment, the spindle checkpoint and cytokinesis. Aurora kinases are regulated through phosphorylation, the binding of specific partners and ubiquitin-dependent proteolysis. Several Aurora substrates have been identified and their roles are being elucidated. The deregulation of Aurora kinases impairs spindle assembly, checkpoint function and cell division, causing missegregation of individual chromosomes or polyploidization accompanied by centrosome amplification. Aurora kinases are frequently overexpressed in cancers and the identification of Aurora A as a cancer-susceptibility gene provides a strong link between mitotic errors and carcinogenesis.     

Differential phosphopeptide expression in a benign breast tissue, and triple-negative primary and metastatic breast cancer tissues from the same African-American woman by LC-LTQ/FT-ICR mass spectrometry

African-American women have a higher risk for developing triple-negative breast cancer (TNBC). Lacking the expression of receptors for estrogen and progesterone, and without human epidermal growth factor 2 receptor gene amplification, TNBC is a very aggressive type of breast cancer with a high likelihood of metastasis and recurrence. Specific therapeutic targets for this aggressive disease remain to be identified. Phosphorylation, a post-translational modification that adds one or more phosphate groups to a protein, plays a key role in the activation and deactivation of a protein’s cellular function. Here, we report the first systematic phosphoproteomic analysis of a benign breast tissue, a primary breast cancer tissue, and a metastatic breast cancer tissue from the same African-American woman. Differential phosphoprotein levels were measured with reversed-phase nano-liquid chromatography coupled to a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FT-ICR MS). Five proteins were found to be highly phosphorylated in the metastatic site whereas six proteins were highly phosphorylated in the cancer site of the TNBC patient. Identified phosphoproteins are known to be involved in breast cancer signal transduction pathways and these results may identify new diagnostic and therapeutic targets for TNBC.     

The functional diversity of Aurora kinases: a comprehensive review

Aurora kinases are serine/threonine kinases essential for the onset and progression of mitosis. Aurora members share a similar protein structure and kinase activity, but exhibit distinct cellular and subcellular localization. AurA favors the G2/M transition by promoting centrosome maturation and mitotic spindle assembly. AurB and AurC are chromosome-passenger complex proteins, crucial for chromosome binding to kinetochores and segregation of chromosomes. Cellular distribution of AurB is ubiquitous, while AurC expression is mainly restricted to meiotically-active germ cells. In human tumors, all Aurora kinase members play oncogenic roles related to their mitotic activity and promote cancer cell survival and proliferation. Furthermore, AurA plays tumor-promoting roles unrelated to mitosis, including tumor stemness, epithelial-to-mesenchymal transition and invasion. In this review, we aim to understand the functional interplay of Aurora kinases in various types of human cells, including tumor cells. The understanding of the functional diversity of Aurora kinases could help to evaluate their relevance as potential therapeutic targets in cancer.     

Silencing of Prrx1b suppresses cellular proliferation,migration, invasion and epithelial–mesenchymal transition in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.     

Aurora kinase A stabilizes FOXM1 to enhance paclitaxel resistance in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) has a relatively poor outcome. Acquired chemoresistance is a major clinical challenge for TNBC patients. Previously, we reported that kinase‐dead Aurora kinase A (Aurora‐A) could effectively transactivate the FOXM1 promoter. Here, we demonstrate an additional pathway through which Aurora‐A stabilizes FOXM1 by attenuating its ubiquitin in TNBC. Specifically, Aurora‐A stabilizes FOXM1 in late M phase and early G1 phase of the cell cycle, which promotes proliferation of TNBC cells. Knock‐down of Aurora‐A significantly suppresses cell proliferation in TNBC cell lines and can be rescued by FOXM1 overexpression. We observe that paclitaxel‐resistant TNBC cells exhibit high expression of Aurora‐A and FOXM1. Overexpression of Aurora‐A offers TNBC cells an additional growth advantage and protection against paclitaxel. Moreover, Aurora‐A and FOXM1 could be simultaneously targeted by thiostrepton. Combination of thiostrepton and paclitaxel treatment reverses paclitaxel resistance and significantly inhibits cell proliferation. In conclusion, our study reveals additional mechanism through which Aurora‐A regulates FOXM1 and provides a new therapeutic strategy to treat paclitaxel‐resistant triple‐negative breast cancer.     

Triple-negative breast cancer cells rely on kinase-independent functions of CDK8 to evade NK-cell-mediated tumor surveillance

Triple-negative breast cancer (TNBC) is an aggressive malignant disease that is responsible for approximately 15% of breast cancers. The standard of care relies on surgery and chemotherapy but the prognosis is poor and there is an urgent need for new therapeutic strategies. Recent in silico studies have revealed an inverse correlation between recurrence-free survival and the level of cyclin-dependent kinase 8 (CDK8) in breast cancer patients. CDK8 is known to have a role in natural killer (NK) cell cytotoxicity, but its function in TNBC progression and immune cell recognition or escape has not been investigated. We have used a murine model of orthotopic breast cancer to study the tumor-intrinsic role of CDK8 in TNBC. Knockdown of CDK8 in TNBC cells impairs tumor regrowth upon surgical removal and prevents metastasis. In the absence of CDK8, the epithelial-to-mesenchymal transition (EMT) is impaired and immune-mediated tumor-cell clearance is facilitated. CDK8 drives EMT in TNBC cells in a kinase-independent manner. In vivo experiments have confirmed that CDK8 is a crucial regulator of NK-cell-mediated immune evasion in TNBC. The studies also show that CDK8 is involved in regulating the checkpoint inhibitor programmed death-ligand 1 (PD-L1). The CDK8–PD-L1 axis is found in mouse and human TNBC cells, underlining the importance of CDK8-driven immune cell evasion in these highly aggressive breast cancer cells. Our data link CDK8 to PD-L1 expression and provide a rationale for investigating the possibility of CDK8-directed therapy for TNBC.Subject terms: Breast cancer, Immune evasion     

The role of EMT-related lncRNA in the process of triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) is the most malignant and fatal subtype of breast cancer, which has characterized by negativity expression of ER, PR, and HER2. Metastasis is the main factor affecting the prognosis of TNBC, and the process of metastasis is related to abnormal activation of epithelial–mesenchymal transition (EMT). Recent studies have shown that long non-coding RNA (LncRNA) plays an important role in regulating the metastasis and invasion of TNBC. Therefore, based on the metastasis-related EMT signaling pathway, great efforts have confirmed that LncRNA is involved in the molecular mechanism of TNBC metastasis, which will provide new strategies to improve the treatment and prognosis of TNBC. In this review, we summarized many signal pathways related to EMT involved in the transfer process. The advances from the most recent studies of lncRNAs in the EMT-related signal pathways of TNBC metastasis. We also discussed the clinical research, application, and challenges of LncRNA in TNBC.     

Discovering alkylamide derivatives of bexarotene as new therapeutic agents against triple-negative breast cancer

Triple-negative breast cancer (TNBC) has been reported to be correlated with high expression of proliferation markers as well as constitutive activation of metastasis-relevant signaling pathways. For many years, breast cancer researchers have been investigating specific and effective methods to treat or to control the development of TNBC, but promising therapeutic options remain elusive. In this study, we have demonstrated that alkylamide derivatives of bexarotene DK-1–150 and DK-1–166 induce apoptotic cell death in TNBC cell lines without causing cytotoxicity in the normal mammary epithelial cell line. Furthermore, the bexarotene derivatives also showed significant effects in inhibiting TNBC cell proliferation and migration, modulating cancer stem cell markers expressions, as well as limiting the epithelial-mesenchymal transition (EMT) activities of TNBC cell lines in terms of downregulating EMT marker and blocking nuclear translocation of β-catenin. Therefore, we propose the alkylamide derivatives of bexarotene as potential candidates for novel anticancer therapeutics against TNBC.     

Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A inhibitors

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either enzymatic assays or cellular phenotypic assays, and displaying more potent anti-proliferation compared with that of VX-680. The most potent compound 10a forms better interaction with Aurora A than Aurora B in molecular docking. Mechanistic studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the produced series of compounds are potential anticancer agents for further development as selective Aurora A inhibitors.     

Aurora kinases: structure, functions and their association with cancer

Background: Aurora kinases are a recently discovered family of kinases (A, B & C) consisting of highly conserved serine\threonine protein kinases found to be involved in multiple mitotic events: regulation of spindle assembly checkpoint pathway, function of centrosomes and cytoskeleton, and cytokinesis. Aberrant expression of Aurora kinases may lead to cancer. For this reason the Aurora kinases are potential targets in the treatment of cancer. In this review we discuss the biology of these kinases: structure, function, regulation and association with cancer. Methods and Results: A literature search. Conclusion: Many of the multiple functions of mitosis are mediated by the Aurora kinases. Their aberrant expression can lead to the deregulation of cell division and cancer. For this reason, the Aurora kinases are currently one of the most interesting targets for cancer therapy. Some Aurora kinase inhibitors in the clinic have proven effectively on a wide range of tumor types. The clinical data are very encouraging and promising for development of novel class of structurally different Aurora kinase inhibitors. Hopefully the Aurora kinases will be potentially useful in drug targeted cancer treatment.     

Roles of Aurora kinases in mitosis and tumorigenesis

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis.     

miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling

Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3′-untranslated region (3′-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3′-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.     

Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

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. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.