Nancy D. Ebelt

Jnk2 effects on tumor development, genetic instability and replicative stress in an oncogene-driven mouse mammary tumor model.

Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK) proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2−/− tumors. In vitro, PyV MT/jnk2−/− cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1) and p21Waf1 protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A) coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

c-Jun N-Terminal Kinases Mediate a Wide Range of Targets in the Metastatic Cascade

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion

Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression. DOI: http://dx.doi.org/10.7554/eLife.07270.001

A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

Triple negative breast cancers (TNBC) have poor prognosis compared to other breast cancer subtypes and represent 15-20% of breast cancers diagnosed. Unique targets and new molecularly-targeted therapies are urgently needed for this subtype. Despite high expression of Epidermal Growth Factor Receptor, inhibitors such as lapatinib have not shown therapeutic efficacy in TNBC patients. Herein, we report that treatment with the covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death, while these compounds as single agents have little effect. The combination significantly increases survival of mice bearing xenografts of MDA-MB-231 human TNBC cells. Our studies demonstrate that lapatinib treatment increases c-Jun and JNK phosphorylation indicating a mechanism of resistance. Combined, these compounds significantly reduce transcriptional activity of Nuclear Factor kappa B, Activating Protein 1, and Nuclear factor erythroid 2-Related Factor 2. As master regulators of antioxidant response, their decreased activity induces a 10-fold increase in reactive oxygen species that is cytotoxic, and is rescued by addition of exogenous antioxidants. Over expression of p65 or Nrf2 also significantly rescues viability during JNK-IN-8 and lapatinib treatment. Further studies combining JNK-IN-8 and lapatinib may reveal a benefit for patients with TNBC, fulfilling a critical medical need.

Serotonin Analogues as Inhibitors of Breast Cancer Cell Growth

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

Abstract 3774: KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Development and screening of small molecule compounds for anti-cancer activity has been of prime interest to the scientific community following the success of targeted, large anti-cancer molecules such as therapeutic antibodies. Small molecules pass more easily through cell membranes and may cross the blood-brain barrier. KD06 is a small molecule triptan-like compound whose parent molecule binds and inhibits serotonin receptors. This compound increases apoptosis of the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-157 via caspase activation. Treatment with KD06 also causes increased autophagy as well as activation of ER stress responses. The growth of tumor xenografts of MDA-MB-231 cells in nude mice are significantly inhibited by twice weekly treatment with 30mg/kg KD06. Analysis of signaling changes by KD06 using reverse phase protein array (RPPA) revealed significant decreases in Akt/mTOR signaling leading to decreased activation of the translation initiation factor 4E-BP1. Other notable changes included decreased expression of proteins important for mitosis such as Cyclin B1, Aurora B and PLK1, and increased phosphorylation of EGFR and increased expression of PDGFR. Analysis of PIP3 and ATP levels showed no change after treatment with KD06, indicating that decreased signaling through Akt/mTOR is not likely due to PI3K inhibition or AMPK activation. Immunofluorescence with KD06 treated cells revealed a change in cell shape after 4 hours of treatment that was reminiscent of cells treated with microtubule binding drugs. Akt localization was affected. These results imply that KD06 may have anti-cancer activity through its effect on microtubule dynamics, inhibiting proper localization and signaling of molecules important for survival and protein translation such as Akt and mTOR. Citation Format: Nancy D. Ebelt, Clint D J Taveres, Xuemei Xie, Youssef W. Naguib, Jiney Jose, Tinashe B. Ruwona, Ashwini K. Devkota, Jihyun Park, Tamer S. Kaoud, Eric V. Anslyn, Jeffrey T. Chang, Zhengrong Cui, Chandra Bartholomeusz, Kevin N. Dalby. KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts. [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 3774.

Abstract 3771: Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation

Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Several ERK inhibitors have already entered clinical trials. Most of the available ERK inhibitors are reversible inhibitors that either act through an allosteric mechanism, or by targeting the ATP binding site. Taking advantage of our understanding of ERK-docking interactions we tried to discover an irreversible substrate-selective inhibitor that targets the protein-binding site of ERK. Here, we report the discovery of a covalent inhibitor of ERK that targets its protein-docking site. Protein NMR, Mass spectroscopy, mutagenesis and molecular docking studies indicate a covalent interaction of the inhibitor with a conserved cysteine residue, Cys-159. Extensive biochemical studies provide an estimate of its kinetic parameters and its kinase-selectivity profile. The new ERK inhibitor inhibits ERK activation, as well as its ability to phosphorylate downstream substrates (e.g. p90RSK and Elk-1) in HEK293T and A375 melanoma cells. The targeting of ERK in HEK293T cells was confirmed using a chemical-genetic approach where the ERK2 C159A mutant was used to rescue the effects of this compound on ERK2 signaling and cell proliferation. Currently, we are testing the effect of the compound on tumor growth inhibition in an A375 melanoma cancer xenografts model. This covalent inhibitor represents a potentially valuable lead molecule whose development may result in a novel class of pharmacologically useful ERK inhibitors for targeting resistant forms of melanoma. Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Mangalika Warthaka, Micael Cano, Rachel Sammons, Qiantao Wang, Pengyu Ren, Ranajeet Ghose, Kevin N. Dalby. Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation. [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 3771.

Abstract P6-04-17: The irreversible c-Jun N-terminal kinase (JNK) inhibitor, JNK-IN-8, sensitizes basal-like breast cancer cells to lapatinib

Basal-like breast cancers represent 15-20% of breast cancers diagnosed each year. These tumors appear earlier in life and have the worst prognosis due to high prevalence of metastasis. Endocrine and molecularly targeted therapies such as trastuzumab or lapatinib are ineffective against this tumor subtype. A high frequency of p53 mutations, low BRCA1 expression along with high epidermal growth factor receptor (EGFR) expression provide some insight into what types of targeted therapies may be useful for basal-like tumors in the future. We have recently shown that JNK2 mediates the expression of p53 and BRCA1 in response to EGFR activation to promote epithelial to mesenchymal transition and metastasis. Further, high JNK2 expression is associated with poorer survival in patients with basal-like breast cancer. These findings suggest that the JNK pathway may be a promising target for basal-type breast cancer treatment. Commonly used competitive ATP inhibitors of JNK have suffered from lack of specificity for JNK. However, the Gray laboratory recently developed highly specific covalent inhibitors of JNK (Zhang, et al. 2012). The agent showing the best properties is JNK-IN-8. Our data show that treatment of human and mouse basal-like, breast cancer cell lines with JNK-IN-8 sensitizes them to lapatinib. Single agents had minor affects on cell viability and cell cycle regulation, but combination treatment led to G2/M arrest and endoreduplication along with synergistic apoptotic responses. Further, the expression and electrophoretic mobility of mitotic arrest deficient 2 (MAD2) (a protein mediating chromosomal segregation) decreased after combination treatment which could explain the chromosomal duplication abnormalities observed. Combination treatment using lapatinib and JNK-IN-8 modulates extracellular signal-regulated kinase (ERK) phosphorylation unlike either drug alone suggesting that both JNK and ERK target G2/M check point proteins, and when perturbed, sensitize basal-type breast cancer cells to targeted therapies. We conclude that use of specific JNK inhibitors in tumors that are resistant to lapatinib, or perhaps trastuzumab, may sensitize them to treatments with these drugs, and may also prevent resistance to these drugs in tumors that were initially responsive. Considering that JNKs are nodes in receptor tyrosine kinase and many other signaling pathways, JNK-IN-8 treatment may improve the efficacy of other targeted therapies as well. Zhang, T. et al. “Discovery of potent and selective covalent inhibitors of JNK”. Chem Biol. 2012 Jan 27;19(1):140-54. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-17.

PD08-01: JNK2 Regulates Mammary Lineage Differentiation in Tumors and Normal Glands through Notch1 and p53.

The classification of patient tumors by clinical subtype has gained wide acceptance due to the implications for prognosis and treatment. However, recent studies have cast doubt on previously advocated normal mammary origins of these subtypes. Thus, the link between the normal mammary gland and mammary tumors is more complex than expected. C-Jun N-Terminal Kinase-2 (JNK2) is a protein that is involved in numerous developmental processes and our previous work has shown it to be important for DNA damage response in mammary tumors. In attempt to gain insight into the link between mammary development and tumorigenesis, we compared normal mammary glands of JNK2 knockout (jnk2ko) mice to jnk2ko mammary tumors expressing or lacking wildtype p53 (p53ko). These studies showed that jnk2ko glands possess 35% fewer basal cells (p=0.0078) with a corresponding increase in luminal epithelial cell populations (p=0.100). This luminal response is corroborated by in vitro 3D assays of primary mammary epithelial cells (MECs) where luminal cell differentiation is normalized by inhibition of Notch signaling. Expression notch-1, a well-known regulator of MEC differentiation, is increased jnk2ko mammary glands. Increased expression of the Notch-1 target gene, hes-1, was also seen (p=0.005). Histology revealed that increased expression of active Notch-1 is localized to the mammary stem cell niche, the terminal end bud. Similar to the normal gland, jnk2ko mammary tumors possessing wildtype p53 exhibit decreased proportions of basal cells (p=0.0002) and increased proportions of luminal cells (p=0.0411) relative to wildtype. Jnk2ko cell lines derived from these tumors show decreased expression of notch-1 (p=0.0018) and hes-1 (p=0.0602) following introduction of JNK2. Luciferase assays comparing activity of the notch-1 promoter to a notch-1 promoter with mutated p53 response elements revealed a dependence of increased notch-1 promoter activity in jnk2ko cells on the p53 response element. P53ko tumor cells, by contrast, do not exhibit alterations in notch-1 promoter activity in the absence of p53 response elements, regardless of JNK2 status. QPCR showed that loss of JNK2 in normal mammary glands and tumors causes increased p53 expression—thus providing a potential mechanism. In support that Notch upregulation in the absence of JNK2 is dependent upon p53, normal glands lacking p53 show no differences in lineage differentiation. P53ko tumors also show no differences in basal lineage differentiation, however, increases in luminal differentiation are maintained in the absence of JNK2. Consistent with increased luminal differentiation, jnk2ko caused decreased expression of markers involved in the epithelial to mesenchymal transition phenotype. This data suggests that JNK2 is important not only for lineage differentiation in normal mammary glands, but in mammary tumors and that the effect is dependent on both Notch1 and p53. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-01.

P1-02-01: c-Jun N-Terminal Kinase 1 (JNK1) Inhibits Tumor Growth and Metastasis by Downregulating Epithelial to Mesenchymal Transition (EMT) and Stem Cell-Related Genes.

Murine and human mammary cancers often show dysregulation of important signaling pathways including the canonical Wingless-iNTegrated (Wnt) and avian ERythroBlastosis oncogene B (ErbB) pathways. Transgenic expression of Wnt ligands causes transformation of normal mammary cells in mice, and Wnt10b is frequently upregulated in human breast cancers. Overexpression of ErbB ligands and amplification of receptors have also been implicated in human breast cancer. JNK1 is a tumor suppressor in the skin and intestinal epithelium, and JNKs are known to integrate ErbB and Wnt pathways as well as others to control cell growth and differentiation. In a murine mammary cancer model where 4T1.2 cells were injected into the mammary gland, reducing JNK1 levels by expressing shRNA (shJNK1) resulted in increased tumor growth and lung metastasis compared to mice injected with control vector- (pSM2) expressing cells. A microarray analysis comparing gene expression between shJNK1 and control tumors revealed 2 and 2.5-fold increases in the ErbB pathway genes Nrg3 and ETV5, 2-fold increases in the Wnt genes Bcl-9 and Wnt10a, and a 1.6-fold increase in the EMT gene Twist1. RT-PCR analysis of in vitro grown 4T1.2 cell lines transfected with shJNK1 confirmed increased expression of Bcl-9 and ETV5, as well as the ETV5 target Cox-2. ErbB2 protein was also overexpressed. The shJNK1 cells showed upregulation of pERK in response to Heregulin1 (an Erbb2/3 ligand) and Fibroblast Growth Factor (FGF) 1, which further amplifies canonical Wnt signaling. In a p53-/− tumor model, ETV5, Bcl-9, and Cox-2 were still upregulated in jnk1-/−compared to wildtype tumor cells, indicating this effect is p53-independent. In the normal mammary gland, a 4-fold increase in ETV5 and a 5-fold increase in Twist1 were found in jnk1-/− mice compared to wildtype, further indicating that this effect is dependent on JNK1 alone. The ErbB and Wnt pathways are known to upregulate EMT and stem-cell related genes, however, the involvement of JNK1 in these effects is a novel hypothesis. Thus far, our data suggest that JNK1 deficiency targets these oncogenic pathways to contribute to a more aggressive tumor phenotype due to heightened EMT and “stem-cellness”. Further studies using cell sorting and differentiation assays will determine whether normal jnk1-/− glands contain a higher fraction of stem cells than wildtype glands. Inhibition of EMT genes and/or ETV5 in the p53-/−;jnk1-/− cancer cells will determine whether the tumor growth or metastasis phenotypes are dependent on these genes. EMT and stem-cell genes are frequently expressed in human breast cancer subtypes that exhibit low survival rates, and EMT is known to be linked to increased metastasis. Some of these sub-types, such as claudin-low tumors, currently have no molecularly-targeted treatments, therefore it is important to determine what proteins critically contribute to these phenotypes so that efficient and effective treatments can be developed. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-01.