Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

Targeting PML in triple negative breast cancer elicits growth suppression and senescence.

Oncogene addiction postulates that the survival and growth of certain tumor cells is dependent upon the activity of one oncogene, despite their multiple genetic and epigenetic abnormalities. This phenomenon provides a foundation for molecular targeted therapy and a rationale for oncogene-based stratification. We have previously reported that the Promyelocytic Leukemia protein (PML) is upregulated in triple negative breast cancer (TNBC) and it regulates cancer-initiating cell function, thus suggesting that this protein can be therapeutically targeted in combination with PML-based stratification. However, the effects of PML perturbation on the bulk of tumor cells remained poorly understood. Here we demonstrate that TNBC cells are addicted to the expression of this nuclear protein. PML inhibition led to a remarkable growth arrest combined with features of senescence in vitro and in vivo. Mechanistically, the growth arrest and senescence were associated to a decrease in MYC and PIM1 kinase levels, with the subsequent accumulation of CDKN1B (p27), a trigger of senescence. In line with this notion, we found that PML is associated to the promoter regions of MYC and PIM1, consistent with their direct correlation in breast cancer specimens. Altogether, our results provide a feasible explanation for the functional similarities of MYC, PIM1, and PML in TNBC and encourage further study of PML targeting strategies for the treatment of this breast cancer subtype.

PGC1α Suppresses Prostate Cancer Cell Invasion through ERRα Transcriptional Control.

The PPARγ coactivator 1 alpha (PGC1α) is a prostate tumor suppressor that controls the balance between anabolism and catabolism. PGC1A downregulation in prostate cancer is causally associated with the development of metastasis. Here we show that the transcriptional complex formed by PGC1α and estrogen-related receptor 1 alpha (ERRα) controls the aggressive properties of prostate cancer cells. PGC1α expression significantly decreased migration and invasion of various prostate cancer cell lines. This phenotype was consistent with remarkable cytoskeletal remodeling and inhibition of integrin alpha 1 and beta 4 expression, both in vitro and in vivo. CRISPR/Cas9-based deletion of ERRα suppressed PGC1α regulation of cytoskeletal organization and invasiveness. Mechanistically, PGC1α expression decreased MYC levels and activity prior to inhibition of invasiveness. In addition, PGC1α and ERRα associated at the MYC promoter, supporting the inhibitory activity PGC1α. The inverse correlation between PGC1α-ERRα activity and MYC levels was corroborated in multiple prostate cancer datasets. Altogether, these results support that PGC1α-ERRα functions as a tumor-suppressive transcriptional complex through the regulation of metabolic and signaling events. SIGNIFICANCE: These findings describe how downregulation of the prostate tumor suppressor PGC1 drives invasiveness and migration of prostate cancer cells.

Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples. METHODS: A novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in. RESULTS: The 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051). CONCLUSION: The newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions.

Integrative analysis of transcriptomics and clinical data uncovers the tumor-suppressive activity of MITF in prostate cancer.

The dysregulation of gene expression is an enabling hallmark of cancer. Computational analysis of transcriptomics data from human cancer specimens, complemented with exhaustive clinical annotation, provides an opportunity to identify core regulators of the tumorigenic process. Here we exploit well-annotated clinical datasets of prostate cancer for the discovery of transcriptional regulators relevant to prostate cancer. Following this rationale, we identify Microphthalmia-associated transcription factor (MITF) as a prostate tumor suppressor among a subset of transcription factors. Importantly, we further interrogate transcriptomics and clinical data to refine MITF perturbation-based empirical assays and unveil Crystallin Alpha B (CRYAB) as an unprecedented direct target of the transcription factor that is, at least in part, responsible for its tumor-suppressive activity in prostate cancer. This evidence was supported by the enhanced prognostic potential of a signature based on the concomitant alteration of MITF and CRYAB in prostate cancer patients. In sum, our study provides proof-of-concept evidence of the potential of the bioinformatics screen of publicly available cancer patient databases as discovery platforms, and demonstrates that the MITF-CRYAB axis controls prostate cancer biology.

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2-/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2-/-mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2-/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2-/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2-/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.