MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

MYC Interacts with the G9a Histone Methyltransferase to Drive Transcriptional Repression and Tumorigenesis.

MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.

MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability.

The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells.

What false-negative rates of non-invasive testing are active surveillance patients and uro-oncologists willing to accept in order to avoid prostate biopsy?

INTRODUCTION: Repeat prostate biopsies in active surveillance patients are associated with significant complications. Novel imaging and blood/urine-based non-invasive tests are being developed to better predict disease grade and volume progression. We conducted a theoretical study to determine what test performance characteristics and costs would a non-invasive test(s) require in order for patients and their physicians to comfortably avoid biopsy. METHODS: Surveys were administered to two populations to determine an acceptable false-negative rate and cost for such test(s). Active surveillance patients were recruited at time of followup in clinic at Princess Margaret Cancer Centre. Physician members of the Society of Urological Oncology were targeted via an online survey. Participants were questioned about their demographics and other characteristics that might influence chosen error rates and cost. RESULTS: 136 patients and 670 physicians were surveyed, with 130 (95.6%) and 104 (15.5%) responses obtained, respectively. A vast majority of patients (90.6%) were comfortable with a non-invasive test(s) in place of biopsy, with 64.8% accepting a false-negative rate of 5-20%. Most physicians (93.3%) were comfortable with a non-invasive test, with 77.9% accepting a rate of 5-20%. Most patients and physicians felt that a cost of less than $1000 per administration would be reasonable. CONCLUSIONS: Most patients/physicians are comfortable with a non-invasive test(s). Although a 5% error rate seems acceptable to many, a substantial subset feels that 99% or higher negative predictive value is required. Thus, a personalized approach with shared decision-making between patients and physicians is essential to optimize patient care in such situations.

MK5 haplodeficiency attenuates hypertrophy and preserves diastolic function during remodeling induced by chronic pressure overload in the mouse heart.

MAPK-activated protein kinase-5 (MK5) is a protein serine/threonine kinase that is activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. The physiological function(s) of MK5 remains unknown. Here, we examined the effect of MK5 haplodeficiency on cardiac function and myocardial remodeling. At 12 wk of age, MK5 haplodeficient mice (MK5+/-) were smaller than age-matched wild-type littermates (MK5+/+), with similar diastolic function but reduced systolic function. Transverse aortic constriction (TAC) was used to induce chronic pressure overload in 12-wk-old male MK5+/- and MK5+/+ mice. Two weeks post-TAC, heart weight-to-tibia length ratios were similarly increased in MK5+/- and MK5+/+ hearts, as was the abundance of B-type natriuretic peptide and ß-myosin heavy chain mRNA. Left ventricular ejection fraction was reduced in both MK5+/+ and MK5+/- mice, whereas regional peak systolic tissue velocities were reduced and isovolumetric relaxation time was prolonged in MK5+/+ hearts but not in MK5+/- hearts. The TAC-induced increase in collagen type 1-α1 mRNA observed in MK5+/+ hearts was markedly attenuated in MK5+/- hearts. Eight weeks post-TAC, systolic function was equally impaired in MK5+/+ and MK5+/- mice. In contrast, the increase in E wave deceleration rate and progression of hypertrophy observed in TAC MK5+/+ mice were attenuated in TAC MK5+/- mice. MK5 immunoreactivity was detected in adult fibroblasts but not in myocytes. MK5+/+, MK5+/-, and MK5-/- fibroblasts all expressed α-smooth muscle actin in culture. Hence, reduced MK5 expression in cardiac fibroblasts was associated with the attenuation of both hypertrophy and development of a restrictive filling pattern during myocardial remodeling in response to chronic pressure overload.NEW & NOTEWORTHY MAPK-activated protein kinase-5 (MK5)/p38-regulated/activated protein kinase is a protein serine/threonine kinase activated by p38 MAPK and/or the atypical MAPKs ERK3 and ERK4. MK5 immunoreactivity was detected in adult ventricular fibroblasts but not in myocytes. MK5 haplodeficiency attenuated the progression of hypertrophy, reduced collagen type 1 mRNA, and protected diastolic function in response to chronic pressure overload.

BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors.

The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks [1]. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the c-MYC oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence MYC interacting proteins, including >30 known binding partners. Putative novel MYC interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodeling complexes, DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase protein chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the MYC interactors identified here, and we validate the previously unreported MYC-CHD8 interaction using both a yeast two hybrid analysis and the proximity-based ligation assay. In sum, we demonstrate that BioID can be utilized to identify bona fide interacting partners for a chromatin-associated protein in vivo. This technique will allow for a much improved understanding of protein-protein interactions in a previously inaccessible biological setting. BIOLOGICAL SIGNIFICANCE: The c-MYC (MYC) oncogene is a transcription factor that plays important roles in cancer initiation and progression. MYC expression is deregulated in more than 50% of human cancers, but the role of this protein in normal cell biology and tumor progression is still not well understood, in part because identifying MYC-interacting proteins has been technically challenging: MYC-containing chromatin-associated complexes are difficult to isolate using traditional affinity purification methods, and the MYC protein is exceptionally labile, with a half-life of only ~30 min. Developing a new strategy to gain insight into MYC-containing protein complexes would thus mark a key advance in cancer research. The recently described BioID proximity-based labeling technique represents a promising new complementary approach for the characterization of protein-protein interactions (PPIs) in cultured cells. Here we report that BioID can also be used to characterize protein-protein interactions for a chromatin-associated protein in tumor xenografts, and present a comprehensive, high confidence in vivo MYC interactome. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

BioID data of c-MYC interacting protein partners in cultured cells and xenograft tumors.

BioID was performed using FlagBirA⁎ (the R118G biotin ligase mutant protein) and FlagBirA⁎-Myc in HEK293 T-REx cells maintained both under standard cell culture conditions and as mouse xenografts. The mass spectrometry dataset acquired in this study has been uploaded to the MassIVE repository with ID: MSV000078518, and consists of 28 ⁎.raw MS files acquired on an Orbitrap Velos instrument, collected in data-dependent mode. iProphet processed MS/MS search results are also included as a reference. This study has been published as "BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors", by Dingar et al. in the Journal of Proteomics, 2014 [1].

MYC phosphorylation at novel regulatory regions suppresses transforming activity.

Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack.

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes.

The E2F4-p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4-p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4-p130-HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4(-/-) hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis.

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart.

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: alpha, beta, gamma, and delta. To date, the prominent isoform in heart has been thought to be p38alpha. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38gamma and p38delta were expressed at protein levels comparable to p38alpha and 38beta, respectively. In the early phase of pressure-overload hypertrophy (1-7 days after constriction of the transverse aorta), the abundance of p38beta, p38gamma and p38delta mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38alpha and p38gamma in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7-28 days after constriction of the transverse aorta), p38gamma immunoreactivity accumulated in the nucleus whereas the distribution of p38alpha remained unaffected. Hence, both p38alpha and p38gamma are prominent p38 isoforms in heart and p38gamma may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.

Characterization of a novel MK3 splice variant from murine ventricular myocardium.

p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.

Characterization of the expression and regulation of MK5 in the murine ventricular myocardium.

MK5, a member of the MAPK-activated protein kinase family, is highly expressed in the heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3'-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains I-VI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting that the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38 alpha, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38 alpha but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.