17 research outputs found
<html>c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting <i>p53/Notch1</i> and <i>breast cancer gene 1</i> expression</html>
Get PDFBreast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis
Jnk2 Effects on Tumor Development, Genetic Instability and Replicative Stress in an Oncogene-Driven Mouse Mammary Tumor Model
Get PDFOncogenes 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
Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas
Get PDFAlthough theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN
c-Jun N-terminal Kinase 2 Regulates Multiple Receptor Tyrosine Kinase Pathways in Mouse Mammary Tumor Growth and Metastasis
No full textc-Jun N-terminal kinase 2 (JNK2) isoforms are transcribed from the jnk2 gene and are highly homologous with jnk1 and jnk3 transcriptional products. JNK proteins mediate cell proliferation, stress response, and migration when activated by a variety of stimuli, including receptor tyrosine kinases (RTKs), but their ability to influence tumor metastasis is ill defined. To evaluate JNK2 in this manner, we used the highly metastatic 4T1.2 mammary tumor cells. Short hairpin RNA expression directed toward JNK2 (shJNK2) decreases tumor cell invasion. In vivo, shJNK2 expression slows tumor growth and inhibits lung metastasis. Subsequent analysis of tumors showed that shJNK2 tumors express lower GRB2-associated binding protein 2 (GAB2). In vitro, knockdown of JNK2 or GAB2 inhibits Akt activation by hepatocyte growth factor (HGF), insulin, and heregulin-1, while phosphorylation of ERK is constitutive and Src dependent. Knockdown of GAB2 phenocopies knockdown of JNK2 in vivo by reducing tumor growth and metastasis, supporting that JNK2 mediates tumor progression by regulating GAB2. The influence of jnk2 in the host or microenvironment was also evaluated using syngeneic jnk2–/– and jnk2+/+ mice. Jnk2–/– mice experience longer survival and less bone and lung metastasis compared to jnk2+/+ mice after intracardiac injection of 4T1.2 cells. GAB2 has previously been shown to mediate osteoclast differentiation, and osteoclasts are critical mediators of tumor-related osteolysis. Thus, studies focusing on the role of JNK2 on osteoclast differentiation were undertaken. ShJNK2 expression impairs osteoclast differentiation, independently of GAB2. Further, shJNK2 4T1.2 cells express less RANKL, a stimulant of osteoclast differentiation. Together, our data support that JNK2 conveys Src/phosphotidylinositol 3-kinase (PI3K) signals important for tumor growth and metastasis by enhancing GAB2 expression. In osteoclast progenitor cells, JNK2 promotes differentiation, which may contribute to the progression of bone metastasis. These studies identify JNK2 as a tumor and host target to inhibit breast cancer growth and metastasis
pH2AX and 53BP1 staining in PyV MT tumors.
No full text<p><b>A</b>). Paraffin embedded PyV MT tumors were stained with pH2AX antibody (green). Nuclei were counter stained with propidium iodide (PI, red); Inset shows high magnification of a cell from a PyV MT/<i>jnk2+/+</i> tumor with arrows pointing at foci. <b>B</b>). Tumor sections were processed as in A) except incubated with 53BP1 primary antibody. Images were captured and pseudo-colored. 53BP1 antibody (red) and DAPI nuclear stain (green). Inset shows high magnification of a cell from the PyV MT/<i>jnk2+/+</i> with arrows pointing at foci. <b>C</b>). Cells with ≥4 nuclear 53BP1 foci were counted as positive cells in each field; a minimum of 5 fields were counted/mouse. Each point represents a tumor average (p = 0.0286).</p
PyV MT/<i>jnk2</i>−/− cells experience replicative stress and increased p21<sup>Waf1</sup> expression.
No full text<p><b>A</b>). Cells were serum starved and then harvested at different time points after 10% FBS stimulation to evaluate CDT1, p21<sup>Waf1</sup>, p-Chk1 (Ser345), and p-p53 (Ser 15) expression by western blot analysis using primary antibodies directed towards the indicated proteins. CDT1 expression at each time point was normalized to GAPDH and graphed for PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cell lines; <b>B</b>). PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cell lines were infected with either adenoviral-GFP or adenoviral-CDT1. Forty-eight hours later, cells were stained using PI with RNase, and then evaluated for cell cycle distribution using flow cytometry; <b>C</b>). Cells were infected with either adenoviral-GFP or adenoviral-CDT1 and harvested 24 hours later. Alternatively, cells were treated with hydroxyurea (HU 5 mM) for 24 hours and then harvested. Western blot analysis was used to measure pChk1 (Ser 345) and p21<sup>Waf1</sup> expression. GAPDH was used to compare sample loading; <b>D</b>). Cells were infected with either adenoviral-GFP or adenoviral-CDT1 during 24 hours of serum starvation then stimulated with 10% FBS and harvested 24 hours later. pChk1 (Ser 345), p-p53 (Ser15), and p21<sup>Waf1</sup> expression was evaluated using western blot analysis. GAPDH was used to compare sample loading.</p
PyV MT/<i>jnk2</i>−/− cellular response is specific to replication and reversed by ATM/ATR inhibitor caffeine.
No full text<p><b>A</b>). PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cells were treated with doxorubicin at the indicated concentrations for 18 hours and then lysed. Expression of p21<sup>Waf1</sup>, p-p53 (Ser15), pH2AX (Ser139), and cleaved caspase 3 were evaluated using western blot analysis. GAPDH was used to compare even loading amongst samples; <b>B</b>). Cells were treated as described in A). except caffeine 2 mM was added as indicated; <b>C</b>). Cells were treated as described in A). except caffeine 2 and 10 mM were added as indicated. Expression of p21<sup>Waf1</sup>, p-p53 (Ser 15), and p53 were evaluated using western blot analysis. GAPDH was used to compare even loading amongst samples.</p
Serum treatment of G<sub>1</sub> arrested cells induces cell death in PyV MT/<i>jnk2</i>−/− cells.
No full text<p><b>A</b>). PyVMT/<i>jnk2+/+</i> and PyVMT<i>/jnk2−/−</i> cells were serum starved for 24 hours and then treated with 10% FBS containing medium. Serum stimulated cells were pulsed with BrdU two hours prior to harvesting and then stained with BrdU primary antibody followed by BrdU detection using flow cytometry. BrdU positivity data are presented as percent positive cells in total cell population; <b>B</b>). PyVMT/<i>jnk2+/+</i> and PyVMT<i>/jnk2−/−</i> cells were serum starved for 24 hours and then treated with 10% FBS containing medium. After 24 hours of serum starvation, cells were either cultured in fresh SFM or medium containing 10% FBS and harvested 24 hours later. Cells were evaluated for Annexin positivity using flow cytometry. Data are expressed as percent positive cells of the total population; <b>C</b>). Cells were serum starved as above and then harvested at indicated time points after 10% FBS stimulation to assess expression of various cell cycle associated proteins using western blot analysis with primary antibodies directed towards the indicated proteins. GAPDH was used to compare even sample loading.</p
Genes of interest on chromosomal segments.
No full text<p>Genes of interest on chromosomal segments.</p
Systemic <i>jnk2</i> deletion enhances tumor development.
No full text<p>PyV MT<i>/jnk2+/+</i> (n = 12), PyV MT/<i>jnk2+/−</i> (n = 16), and PyV MT/<i>jnk2−/−</i> (n = 19) mice were palpated for mammary tumors thrice weekly. Once palpated, tumor growth was recorded thrice weekly. <b>A</b>). Kaplan Meier graph showing age of first tumor palpation (median age was day 55 for PyV MT<i>/jnk2−/−</i> vs. day 70 for PyV MT<i>/jnk2+/+</i>, p = .11); <b>B</b>). Total number of tumors palpated per mouse at the time of harvest was higher in PyV MT/<i>jnk2−/−</i> mice compared to PyV MT/<i>jnk2+/+</i> mice, p = 0.0192); <b>C</b>). Paraffin embedded, non-target tumor sections were probed with cleaved caspase 3 primary antibody and detected using FITC labeled secondary antibody. Nuclei were stained with propidium iodide. The total number of cells staining positive for cleaved caspase 3 were scored and divided by the total number of nuclei (n = 5 tumors in each group); <b>D</b>). Paraffin embedded tissue sections were probed with Ki-67 primary antibody and detected using DAB. Cells staining positive for Ki-67 were counted and divided by the total number of nuclei (Hematoxylin) per field. Five fields per tumor were counted (n = 5 per genotype, p = 0.0159); <b>E</b>). Paraffin embedded tissue sections were probed with p-c-Jun (Ser63) primary antibody and detected using DAB. Hematoxylin was used as a nuclear stain.</p
