Endocrine resistance and metastatic progression are primary causes of treatment failure in breast cancer. While mitogen activated protein kinases (MAPKs) are known to promote ligand-independent cell growth, the role of the MEK5-ERK5 pathway in the progression of clinical breast carcinoma remains poorly understood. Here, we demonstrated increased ERK5 activation in 30 of 39 (76.9%) clinical tumor samples, as well as across breast cancer cell systems. Overexpression of MEK5 in MCF-7 cells promoted both hormone-dependent and hormone-independent tumorigenesis in vitro and in vivo and conferred endocrine therapy resistance to previously sensitive breast cancer cells. Expression of MEK5 suppressed estrogen receptor (ER)α, but not ER-β protein levels, and abrogated downstream estrogen response element (ERE) transcriptional activity and ER-mediated gene transcription. Global gene expression changes associated with upregulation of MEK5 included increased activation of ER-α independent growth signaling pathways and promotion of epithelial-to-mesenchymal transition (EMT) markers. Taken together, our findings show that the MEK5-ERK5 pathway mediates progression to an ER(−), mesenchymal and endocrine therapy resistant phenotype. Given the need for new clinical therapeutic targets, our results demonstrate the therapeutic potential of targeting the MEK5-ERK5 pathway in breast cancer.
The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E2). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gαo protein subunit potentiated ERα activity in the absence and presence of E2. Transient transfection of the human breast cancer cell line MCF-7 showed that Gαo augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gαo revealed that Gαo stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gαo, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.
Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-α (TNFα) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFα. Our gene reporter assays revealed that ERα, not ERβ, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERα for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERα and ERβ as well as regulation of GRIP1.
estrogen receptor; breast cancer; AKT; cell signaling; cell survival
Acquired chemoresistance and epithelial-to-mesenchymal transition (EMT) are hallmarks of cancer progression and of increasing clinical relevance. We investigated the role of miRNA and p38 mitogen-activated protein kinase (MAPK) signaling in the progression of breast cancer to a drug-resistant and mesenchymal phenotype. We demonstrate that acquired death receptor resistance results in increased hormone-independent tumorigenesis compared to hormone-sensitive parental cells. Utilizing global miRNA gene expression profiling, we identified miRNA alterations associated with the development of death receptor resistance and EMT progression. We further investigated the role of p38 MAPK in this process, showing dose-dependent inactivation of p38 by its inhibitor RWJ67657 and decreased downstream ATF and NF-κB signaling. Pharmacological inhibition of p38 also decreased chemoresistant cancer tumor growth in xenograft animal models. Interestingly, inhibition of p38 partially reversed the EMT changes found in this cell system, as illustrated by decreased gene expression of the EMT markers Twist, Snail, Slug and ZEB and protein and mRNA levels of Twist, a known EMT promoter, concomitant with decreased N-cadherin protein. RWJ67657 treatment also altered the expression of several miRNAs known to promote therapeutic resistance, including miR-200, miR-303, miR-302, miR-199 and miR-328. Taken together, our results demonstrate the roles of multiple microRNAs and p38 signaling in the progression of cancer and demonstrate the therapeutic potential of targeting the p38 MAPK pathway for reversing EMT in an advanced tumor phenotype.
p38 mitogen-activated protein kinase; epithelial-tomesenchymal transition; breast cancer; drug discovery
The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)-CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA-MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA-MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA-MB-361 and MCF-7-CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA-MB-361 and MCF-7-CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1-CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.
SDF-1; CXCR4; microRNA; breast carcinoma; hormone independence; AMD3100
Endocrine therapy resistance is a primary cause of clinical breast cancer treatment failure. The p38 mitogen activated protein kinase (MAPK) signaling pathway is known to promote ligand independent tumor growth and resistance to endocrine therapy. In this study, we investigated the therapeutic potential of the p38 inhibitor RWJ67657 in the treatment of tamoxifen resistant MDA-MB-361 cells. RWJ67657 dose-dependently decreased both basal and stimulated activation of p38 MAPK signaling in this drug resistant cell system. Decreased activation of p38 by RWJ67657 resulted in inhibition of the downstream p38 targets hsp27 and MAPKAPK. Diminished p38 signaling resulted in inhibition of p38-medated gene transcription. Furthermore, pharmacological inhibition of p38 by RWJ67657 decreased biological effects of p38, including ER-mediated gene expression and clonogenic survival in a dose-dependent manner. Animal studies revealed significantly decreased p38 signaling in vivo following exposure to RWJ67657. Treatment with the inhibitor markedly decreased phosphorylation of p38 in MDA-MB-361 tumors, leading to decreased transcription of both Fra-1 and progesterone receptor. Utilizing well-established xenograft tumor models, we demonstrated that RWJ67657 exhibits potent anti-tumor properties. Treatment with RWJ67657 markedly decreased tamoxifen resistant tumor growth, both in the presence and absence of estrogen. Taken together, our findings demonstrate the therapeutic potential of targeting the p38-MAPK signaling cascade in the treatment of endocrine resistant breast cancer.
p38; mitogen-activated protein kinase; endocrine resistance; breast cancer; drug discovery; cancer biology; hormone independence; kinase inhibitors; estrogen receptor; gene transcription
Resistance to chemotherapy remains a significant obstacle in the treatment of hormone-independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC50 concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NFκB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemoand endocrine therapy-resistant breast cancer.
sphingolipids; chemoresistance; sphingosine kinase; NFkappaB; breast cancer; ceramide; TNF; sphingosine-1-phosphate
Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).
We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.
Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.
Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.
Congenital chest wall deformities are considered to be anomalies in chest wall growth. These can be categorized as either rib cage overgrowth or deformities related to inadequate growth (aplasia or dysplasia). Rib cage overgrowth leads to depression of the sternum (pectus excavatum) or protuberance of the sternum (pectus carinatum) and accounts for greater than 90% of congenital chest wall deformities. The remaining deformities are a result of inadequate growth. Evolution in the management of congenital chest wall deformities has made significant progress over the past 25 years. This article will review chest wall deformities and the current management strategies of these interesting anomalies.
Congenital; chest wall; deformity; pectus
Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1–CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1–CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1–mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.
Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into antiestrogenic ligands by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of daidzein analogues have been designed and synthesized. When MCF-7 cells were treated with the analogues, those resulting from hydrogen substitution by isopropyl (3d), isobutyl (3f), cyclopentyl (3g), and pyrano- (2), inhibited cell proliferation, estrogen-induced transcriptional activity, and estrogen receptor (ER) regulated progesterone receptor (PgR) gene expression. However, methyl (3a) and ethyl (3b) substitutions of the hydroxyl proton only led to moderate reduction of the estrogenic activities. These results demonstrated the structural requirements for the transformation of daidzein from an ER agonist to an antagonist. The most effective analogue, 2 was found to reduce in vivo estrogen stimulated MCF-7 cell tumorigenesis using a xenograft mouse model.
Daidzein analogues; antiestrogen; isoflavones; breast cancer; phytoestrogen; SERM; estrogen receptor; xenograft model
Adult human mesenchymal stem cells (hMSC) have been shown to home to sites of carcinoma and affect biological processes, including tumour growth and metastasis. Previous findings have been conflicting and a clear understanding of the effects of hMSCs on cancer remains to be established. Therefore, we set out to investigate the impact of hMSCs on the oestrogen receptor positive, hormone-dependent breast carcinoma cell line MCF-7.
In this study, we show the effects of hMSCs on cancer cells are mediated through a secreted factor(s) which are enhanced by cancer cell-hMSC contact/communication. In addition to enhanced proliferation when in co-culture with hMSCs, MCF-7 cells were found to have increased migration potential in vitro. Inhibition of ER signalling by the pure anti-oestrogen ICI 182,780 decreased the effect of hMSCs on MCF-7 cell proliferation and migration supporting a role for ER signalling in the hMSC/MCF-7 cell interaction. Additionally, hMSCs have been shown to secrete a wide variety of growth factors and chemokines including stromal cell-derived factor-1 (SDF-1). This coupled with the knowledge that SDF-1 is an ER-mediated gene linked with hormone-independence and metastasis led to the investigation of the SDF-1/CXCR4 signalling axis in hMSC-MCF-7 cell interaction. Experiments revealed an increase in SDF-1 gene expression both in vivo and in vitro when MCF-7 cells were cultured with hMSCs. SDF-1 treatment of MCF-7 cells alone increased proliferation to just below that seen with hMSC co-culture. Additionally, blocking SDF-1 signalling using a CXCR4-specific inhibitor decreased hMSC induced proliferation and migration of MCF-7. However, the combined treatment of ICI and AMD3100 reduced MCF-7 cell proliferation and migration below control levels, indicating targeting both the ER and CXCR4 pathways is effective in decreasing the hMSCs induction of MCF-7 cell proliferation and migration.
The sum of these data reveals the relationship between tumour microenvironment and tumour growth and progression. Better understanding of the mechanisms involved in this tumour stroma cell interaction may provide novel targets for the development of treatment strategies for oestrogen receptor positive, hormone-independent, and endocrine-resistant breast carcinoma.
Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production.
Methods and Results
Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-β subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, P=0.004) and specific activity (+20%, P=0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity.
CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function.
cardiac resynchronization therapy; mitochondria; proteomics; ATP synthase
The glycosylation state of envelope glycoproteins in Human and Simian Immunodeficiency Viruses (HIV/SIV) is critical to viral infectivity and tropism, viral protein processing, and in virus evasion of the immune system. Using a rapid fluorescent two-dimensional gel based method coupled with enzymatic pre-treatment of virus with PNGase F (Peptide: N-Glycosidase F) and fluorescent 2D gels or 2D gel Western blotting, we show significant differences in the glycosylation patterns of two SIV strains widely used in animal models of HIV disease and vaccine studies. We also demonstrate the modification of a host protein important in HIV biology (HLA-DR) by O-GlcNAc. Further, this experimental pipeline allows for the identification of the modified protein and the site of N-linked glycosylation by fluorescent two-dimensional gel electrophoresis coupled with mass spectrometry (MS) and the qualitative and semi-quantitative assessment of viral glycosylation. The method is fully compatible with downstream glycomics analysis. This approach will permit correlation of virus glycosylation status with pathological severity and may serve as a rapid screen of viruses from physiological samples for further study by more advanced MS methodology.
HIV-1; SIV; Glycosylation; O-GlcNAc; N-Linked Glycans; Mass Spectrometry; Proteomics; Two-Dimensional Gels; DIGE; Fluorescence
Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.
The loss of normal anatomic barriers in neonates with congenital diaphragmatic hernia (CDH) can predispose children to gastroesophageal reflux (GER). In an attempt to improve post-operative feeding, we have added a modified anterior fundoplication to restore natural gastric and esophageal positioning.
The institutional review board of both participating centers approved this study. Between 1997 and 2008, 13 neonates with high-risk anatomy underwent repair of CDH combined with an anterior fundoplication (Boix-Ochoa). The anatomic indications for concomitant fundoplication were absence of an intra-abdominal esophagus, an obtuse angle of His, and a small, vertically oriented stomach.
Ten patients survived to discharge and eight were on full oral nourishment. One required partial gastrostomy feedings for an improving oral aversion and quickly progressed to full oral feedings. One patient with chromosomal anomalies and swallowing dysfunction remained on long-term bolus gastrostomy feedings. Two with progressive symptoms of GER and failure to thrive required conversion to a 360° wrap after 18 months of medical management. This was performed in conjunction with a planned, staged muscle flap reconstruction in one patient. There were no complications related to the fundoplication.
Anatomic predictors of severe GER can be efficiently countered at the time of CDH repair. A modified fundoplication should be considered in the operative management of high-risk infants.
Reflux; Neonate; CDH; Fundoplication
Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.
To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.
Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.
This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.
The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1.
In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death.
Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.