Kinesin Facilitates Phenotypic Targeting of Therapeutic Resistance in Advanced Prostate Cancer.

Understanding the mechanisms underlying resistance is critical to improving therapeutic outcomes in patients with metastatic castration-resistant prostate cancer (mCRPC). Previous work showed dynamic interconversions between epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) defines the phenotypic landscape of prostate tumors, as a potential driver of emergence of therapeutic resistance. In this study, we use in vitro and in vivo preclinical MDA PCa PDX models of resistant human prostate cancer to determine molecular mechanisms of cross-resistance between anti-androgen therapy and taxane chemotherapy, underlying the therapeutically resistant phenotype. Transcriptomic profiling revealed that resistant and sensitive prostate cancer C4-2B cells have a unique differential gene signature response to cabazitaxel. Gene pathway analysis showed that sensitive cells exhibit increase in DNA damage, while resistant cells express genes associated with protein regulation in response to cabazitaxel. These PDX specimens are from patients who have metastatic lethal CRPC, treated with androgen-deprivation therapy (ADT), antiandrogens and chemotherapy including 2nd line taxane chemotherapy, cabazitaxel. Immunohistochemistry revealed high expression of E-cadherin and low expression of vimentin resulting in re-differentiation toward an epithelial phenotype. Furthermore, the mitotic kinesin-related protein (HSET) involved in microtubule binding and the SLCO1B3 transporter (implicated in cabazitaxel intracellular transport), associated with resistance in these prostate tumors. Combinational targeting of kinesins (ispinesib) with cabazitaxel was more effective than single monotherapies in inducing cell death in resistant prostate tumors. Implications: Our findings are of translational significance in identifying kinesin as a novel target of cross-resistance, towards enhancing therapeutic vulnerability and improved clinical outcomes in patients with advanced prostate cancer.

Activation of STING in Response to Partial-Tumor Radiation Exposure.

PURPOSE: To determine the mechanisms involved in partial volume radiation therapy (RT)-induced tumor response. METHODS AND MATERIALS: We investigated 67NR murine orthotopic breast tumors in Balb/c mice and Lewis lung carcinoma (LLC cells; WT, Crispr/Cas9 Sting KO, and Atm KO) injected in the flank of C57Bl/6, cGAS, or STING KO mice. RT was delivered to 50% or 100% of the tumor volume using a 2 × 2 cm collimator on a microirradiator allowing precise irradiation. Tumors and blood were collected at 6, 24, and 48 hours post-RT and assessed for cytokine measurements. RESULTS: There is a significant activation of the cGAS/STING pathway in the hemi-irradiated tumors compared with control and to 100% exposed 67NR tumors. In the LLC model, we determined that an ATM-mediated noncanonical activation of STING is involved. We demonstrated that the partial exposure RT-mediated immune response is dependent on ATM activation in the tumor cells and on the STING activation in the host, and cGAS is dispensable. Our results also indicate that partial volume RT stimulates a proinflammatory cytokine response compared with the anti-inflammatory profile induced by 100% tumor volume exposure. CONCLUSIONS: Partial volume RT induces an antitumor response by activating STING, which stimulates a specific cytokine signature as part of the immune response. However, the mechanism of this STING activation, via the canonical cGAS/STING pathway or a noncanonical ATM-driven pathway, depends on the tumor type. Identifying the upstream pathways responsible for STING activation in the partial RT-mediated immune response in different tumor types would improve this therapy and its potential combination with immune checkpoint blockade and other antitumor therapies.

Anoikis in phenotypic reprogramming of the prostate tumor microenvironment.

Prostate cancer is one of the most common malignancies in males wherein 1 in 8 men are diagnosed with this disease in their lifetime. The urgency to find novel therapeutic interventions is associated with high treatment resistance and mortality rates associated with castration-resistant prostate cancer. Anoikis is an apoptotic phenomenon for normal epithelial or endothelial cells that have lost their attachment to the extracellular matrix (ECM). Tumor cells that lose their connection to the ECM can die via apoptosis or survive via anoikis resistance and thus escaping to distant organs for metastatic progression. This review discusses the recent advances made in our understanding of the signaling effectors of anoikis in prostate cancer and the approaches to translate these mechanistic insights into therapeutic benefits for reducing lethal disease outcomes (by overcoming anoikis resistance). The prostate tumor microenvironment is a highly dynamic landscape wherein the balance between androgen signaling, cell lineage changes, epithelial-mesenchymal transition (EMT), extracellular matrix interactions, actin cytoskeleton remodeling as well as metabolic changes, confer anoikis resistance and metastatic spread. Thus, these mechanisms also offer unique molecular treatment signatures, exploitation of which can prime prostate tumors to anoikis induction with a high translational significance.

Endothelial mechanisms for inactivation of inflammation-induced hyperpermeability.

Microvascular hyperpermeability is a hallmark of inflammation. Many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function. Therefore, we propose that targeted therapeutic approaches focusing on mechanisms that terminate hyperpermeability would avoid the negative effects of prolonged hyperpermeability while retaining its short-term beneficial effects. We tested the hypothesis that inflammatory agonist signaling leads to hyperpermeability and initiates a delayed cascade of cAMP-dependent pathways that causes inactivation of hyperpermeability. We applied platelet-activating factor (PAF) and vascular endothelial growth factor (VEGF) to induce hyperpermeability. We used an Epac1 agonist to selectively stimulate exchange protein activated by cAMP (Epac1) and promote inactivation of hyperpermeability. Stimulation of Epac1 inactivated agonist-induced hyperpermeability in the mouse cremaster muscle and in human microvascular endothelial cells (HMVECs). PAF induced nitric oxide (NO) production and hyperpermeability within 1 min and NO-dependent increased cAMP concentration in about 15-20 min in HMVECs. PAF triggered phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in a NO-dependent manner. Epac1 stimulation promoted cytosol-to-membrane eNOS translocation in HMVECs and in myocardial microvascular endothelial (MyEnd) cells from wild-type mice, but not in MyEnd cells from VASP knockout mice. We demonstrate that PAF and VEGF cause hyperpermeability and stimulate the cAMP/Epac1 pathway to inactivate agonist-induced endothelial/microvascular hyperpermeability. Inactivation involves VASP-assisted translocation of eNOS from the cytosol to the endothelial cell membrane. We demonstrate that hyperpermeability is a self-limiting process, whose timed inactivation is an intrinsic property of the microvascular endothelium that maintains vascular homeostasis in response to inflammatory conditions.NEW & NOTEWORTHY Termination of microvascular hyperpermeability has been so far accepted to be a passive result of the removal of the applied proinflammatory agonists. We provide in vivo and in vitro evidence that 1) inactivation of hyperpermeability is an actively regulated process, 2) proinflammatory agonists (PAF and VEGF) stimulate microvascular hyperpermeability and initiate endothelial mechanisms that terminate hyperpermeability, and 3) eNOS location-translocation is critical in the activation-inactivation cascade of endothelial hyperpermeability.

Chemotherapeutic Agents-Induced Ceramide-Rich Platforms (CRPs) in Endothelial Cells and Their Modulation.

The prevailing mechanism of action of chemotherapeutic drugs has been challenged by the role of ceramide, a second messenger, shown to induce apoptosis, differentiation, growth arrest, senescence, and autophagy in different cells (Chabner BA, Roberts TG Jr, Nat Rev Cancer 5:65-72, 2005; Jacobi J et al, Cell Signal 29:52-61, 2017; Rotolo J et al, J Clin Invest 122:1786-1790, 2012; Truman JP et al, PLoS One 5:e12310, 2010). Certain chemotherapeutic drugs activate the acid sphingomyelinase (ASMase)/ceramide pathway, generating ceramide in the tumor endothelium and this microvascular dysfunction is crucial for the tumor response. Ceramide has fusigenic properties and as such, when generated within the plasma membrane, initiates the oligomerization of ceramide-and cholesterol-rich domains in the outer leaflet of the plasma membrane, leading to the formation of ceramide-rich microdomains/platforms (CRP) (Jacobi J et al, Cell Signal 29:52-61, 2017; Truman JP et al, PLoS One 5:e12310, 2010; van Hell AJ et al, Cell Signal 34:86-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216:115-130, 2013) known as "signaling platform." This chapter will discuss the generation, detection, and quantitation of CRP and their possible modulation in endothelial cells, in vitro and in vivo in response to certain chemotherapeutic drugs.

Reduced activin receptor-like kinase 1 activity promotes cardiac fibrosis in heart failure.

INTRODUCTION: Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFß1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. HYPOTHESIS: We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. METHODS AND RESULTS: In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1+/-) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1+/- mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and ß-myosin heavy chain were increased similarly in Alk1+/- and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 collagen mRNA and protein levels were higher in Alk1+/- mice. LV fractional shortening was lower in Alk1+/- mice after TAC. CONCLUSIONS: Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms.

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that 1) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, 2) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and 3) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that 1) different hyperpermeability mechanisms operate after brief and prolonged ischemia and 2) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury.NEW & NOTEWORTHY Here, we demonstrate that 1) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells; 2) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and 3) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFß) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n = 5) and vehicle treated AlK-1-cKO mice (cKO-CON; n = 5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n = 15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p < 0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFß-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFß1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of ß-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of AlK-1 in cardiac remodeling independent of systemic AVMs are required.