Purpose of ReviewThis review aims to highlight the past and more current literature related to the multifaceted pathogenic programs that contribute to calcific aortic valve disease (CAVD) with a focus on the contribution of developmental programs.Recent FindingsCalcification of the aortic valve is an active process characterized by calcific nodule formation on the aortic surface leading to a less supple and more stiffened cusp, thereby limiting movement and causing clinical stenosis. The mechanisms underlying these pathogenic changes are largely unknown, but emerging studies have suggested that signaling pathways common to valvulogenesis and bone development play significant roles and include Transforming Growth Factor-β (TGF-β), bone morphogenetic protein (BMP), Wnt, Notch, and Sox9.SummaryThis comprehensive review of the literature highlights the complex nature of CAVD but concurrently identifies key regulators that can be targeted in the development of mechanistic-based therapies beyond surgical intervention to improve patient outcome.

Amplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site-1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del190–515 variant (equivalent to previously reported) expressed in >90% of advanced stage serous epithelial ovarian cancers. Although EVI1Del190–515 lacks ∼70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGFβ signaling, similar to wild type EVI1. This contrasts with EVI1 1–268 which failed to interact with CtBP1. Interestingly, the EVI1Del190–515 splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1Del427–515. While wild type EVI1 efficiently repressed TGFβ-mediated AP-1 (activator protein-1) and plasminogen activator inhibitor-1 (PAI-1) promoters, EVI1Del190–515 elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1Del427–515 (but not EVI1Del190–515) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1Del190–515) markedly increased claudin-1 mRNA and protein expression in HEY ovarian and MDA-MB-231 breast cancer cells. Changes in claudin-1 were associated with alterations in specific epithelial–mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.

SnoN/SkiL (TGFβ regulator) is dysregulated in ovarian cancer, a disease associated with acquired drug-resistance. Arsenic trioxide (As2O3used in treating APL) induces SnoN to oppose the apoptotic response in ovarian cancer cells. We now report that As2O3 increases phosphorylation of EGFR/p66ShcA and EGFR degradation. As2O3 activates Src(Y416) whose activity (inhibited by PP2) modulates EGFR activation, its interaction with Shc/Grb2, and p-AKT. Inhibition of PI3K reduces SnoN and cell survival. Although EGFR or MAPK1 siRNA did not alter SnoN expression, As2O3–induced cleaved PARP was reduced together with increased XIAP. Collectively, As2O3 mediates an initial rise in pY-Src(416) to regulate the PI3K/AKT pathway which increases SnoN and cell survival; these early events may counter the cell death response associated with increased pY-EGFR/MAPK activation.

BackgroundmiRNAs can regulate cellular survival in various cancer cell types. Recent evidence implicates the formation of lipid droplets as a hallmark event during apoptotic cell death response. It is presently unknown whether MIR494, located at 14q32 which is deleted in renal cancers, reduces cell survival in renal cancer cells and if this process is accompanied by changes in the number of lipid droplets.Methods769-P renal carcinoma cells were utilized for this study. Control or MIR494 mimic was expressed in these cells following which cell viability (via crystal violet) and apoptotic cell numbers (via Annexin V/PI staining) were assessed. By western blotting, MIR494 cellular responses were validated using MIR494 antagomir and Argonaute 2 siRNA. Transmission electron microscopy (TEM) was performed in MIR494-transfected 769-P cells to identify ultrastructural changes. LipidTOX green neutral lipid staining and cholesterol measurements were conducted to assess accumulation of lipids droplets and total cholesterol levels, respectively, in MIR494 expressing 769-P cells. Indirect immunofluorescence and western analyses were also performed to examine changes in mitochondria organization. Co-transfection of MIR494 mimic with siRNA targeting LC3B and ATG7 was conducted to assess their contribution to formation of lipid droplets in MIR494-expressing cells.ResultsMIR494 expression reduces viability of 769-P renal cancer cells; this was accompanied by increased cleaved PARP (an apoptotic marker) and LC3B protein. Further, expression of MIR494 increased LC3B mRNA levels and LC3B promoter activity (2.01-fold; 50 % increase). Interestingly, expression of MIR494 markedly increased multilamellar bodies and lipid droplets (by TEM and validated by LipidTOX immunostaining) while reducing total cholesterol levels. Via immunocytochemistry, we observed increased LC3B-associated endogenous punctae upon MIR494 expression. In contrast to ATG7 siRNA, knockdown of LC3B reduced the numbers of lipid droplets in MIR494-expressing cells. Our results also identified that MIR494 expression altered the organization of mitochondria which was accompanied by co-localization with LC3B punctae, decreased PINK1 protein, and altered Drp1 intracellular distribution.ConclusionCollectively, our findings indicate that MIR494 reduces cell survival in 769-P renal cancer cells which is accompanied by increased lipid droplet formation (which occurs in a LC3B-dependent manner) and mitochondrial changes.

Rationale: Calcific Aortic Valve Disease (CAVD) affects more than 5.2 million people in the US. The only effective treatment is surgery and this comes with complications and no guarantee of long-term success. Objective: Outcomes from pharmacological initiatives remain unsubstantiated and therefore the aim of this study is to determine if repurposing a selective XPO1 inhibitor drug (KPT-330) is beneficial in the treatment of CAVD. Methods and Results: We show that KPT-330 prevents, attenuates and mitigates calcific nodule formation in heart valve interstitial cells (VICs) in vitro, and prevents CAVD in Klotho -/- mice. Using RNA-sequencing and Mass Spectrometry we show that KPT-330's beneficial effect is mediated by inhibiting nuclear export of the transcription factor C/EBPβ in VICs, leading to repression of canonical Wnt signaling, in part through activation of the Wnt antagonist Axin1, and a subsequent decrease in pro-osteogenic markers and cell viability. Conclusions: Our findings have met a critical need to discover alterative, pharmacological-based therapies in the treatment of CAVD.