New roles for renin and prorenin in heart failure and cardiorenal crosstalk

The renin-angiotensin-aldosterone-system (RAAS) plays a central role in the pathophysiology of heart failure and cardiorenal interaction. Drugs interfering in the RAAS form the pillars in treatment of heart failure and cardiorenal syndrome. Although RAAS inhibitors improve prognosis, heart failure–associated morbidity and mortality remain high, especially in the presence of kidney disease. The effect of RAAS blockade may be limited due to the loss of an inhibitory feedback of angiotensin II on renin production. The subsequent increase in prorenin and renin may activate several alternative pathways. These include the recently discovered (pro-) renin receptor, angiotensin II escape via chymase and cathepsin, and the formation of various angiotensin subforms upstream from the blockade, including angiotensin 1–7, angiotensin III, and angiotensin IV. Recently, the direct renin inhibitor aliskiren has been proven effective in reducing plasma renin activity (PRA) and appears to provide additional (tissue) RAAS blockade on top of angiotensin-converting enzyme and angiotensin receptor blockers, underscoring the important role of renin, even (or more so) under adequate RAAS blockade. Reducing PRA however occurs at the expense of an increase plasma renin concentration (PRC). PRC may exert direct effects independent of PRA through the recently discovered (pro-) renin receptor. Additional novel possibilities to interfere in the RAAS, for instance using vitamin D receptor activation, as well as the increased knowledge on alternative pathways, have revived the question on how ideal RAAS-guided therapy should be implemented. Renin and prorenin are pivotal since these are at the base of all of these pathways

Non-Apoptotic Cell Death Signaling Pathways in Melanoma

Resisting cell death is a hallmark of cancer. Disturbances in the execution of cell death programs promote carcinogenesis and survival of cancer cells under unfavorable conditions, including exposition to anti-cancer therapies. Specific modalities of regulated cell death (RCD) have been classified based on different criteria, including morphological features, biochemical alterations and immunological consequences. Although melanoma cells are broadly equipped with the anti-apoptotic machinery and recurrent genetic alterations in the components of the RAS/RAF/MEK/ERK signaling markedly contribute to the pro-survival phenotype of melanoma, the roles of autophagy-dependent cell death, necroptosis, ferroptosis, pyroptosis, and parthanatos have recently gained great interest. These signaling cascades are involved in melanoma cell response and resistance to the therapeutics used in the clinic, including inhibitors of BRAFmut and MEK1/2, and immunotherapy. In addition, the relationships between sensitivity to non-apoptotic cell death routes and specific cell phenotypes have been demonstrated, suggesting that plasticity of melanoma cells can be exploited to modulate response of these cells to different cell death stimuli. In this review, the current knowledge on the non-apoptotic cell death signaling pathways in melanoma cell biology and response to anti-cancer drugs has been discussed

Trametinib-Resistant Melanoma Cells Displaying MITF^high/NGFR^low/IL-8^low Phenotype Are Highly Responsive to Alternating Periods of Drug Withdrawal and Drug Rechallenge

Despite significant advances in targeted therapies against the hyperactivated BRAFV600/MEK pathway for patients with unresectable metastatic melanoma, acquired resistance remains an unsolved clinical problem. In this study, we focused on melanoma cells resistant to trametinib, an agent broadly used in combination therapies. Molecular and cellular changes were assessed during alternating periods of trametinib withdrawal and rechallenge in trametinib-resistant cell lines displaying either a differentiation phenotype (MITFhigh/NGFRlow) or neural crest stem-like dedifferentiation phenotype (NGFRhigh/MITFlow). Neither drug withdrawal nor drug rechallenge induced cell death, and instead of loss of fitness, trametinib-resistant melanoma cells adapted to altered conditions by phenotype switching. In resistant cells displaying a differentiation phenotype, trametinib withdrawal markedly decreased MITF level and activity, which was associated with reduced cell proliferation capacity, and induced stemness assessed as NGFR-positive cells and senescence features, including IL-8 expression and secretion. All these changes could be reversed by trametinib re-exposure, which emphasizes melanoma cell plasticity. Trametinib-resistant cells displaying a dedifferentiation phenotype were less responsive presumably due to the already low level of MITF, a master regulator of the melanoma phenotype. Considering new directions of the development of anti-melanoma treatment, our study suggests that the phenotype of melanomas resistant to targeted therapy might be a crucial determinant of the selection of second-line therapy for melanoma patients

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies

Melanoma cells grown in EGF(+)bFGF(+) medium respond to changes in microenvironment by employing pro-survival machinery.

<p>(A) DMBC12, DMBC19, DMBC17 and DMBC10 populations grown in EGF(+)bFGF(+) medium were dissociated and one day later (0 h) they were exposed to serum-containing medium. qRT-PCR was used to analyze the mRNA levels of pro-survival genes at indicated time points of medium exchange. Data are presented as the means ± SD from three independent experiments. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. For clarity, *** (<i>p</i><0.001) for MCL-1 in DMBC12 cells after 1 h was not indicated. (B) Cell lysates were prepared at indicated time points after transfer to serum-containing medium, and pro-survival proteins MCL-1, BCL-X_L and BCL-2 were immunoblotted (n = 3). Localization and molecular weight (MW) of marker bands are indicated. β-actin was used as a loading control. Quantification of protein level is shown above the blots.</p

Characteristics of patient-derived cultures used in this study.

<p>(A) Morphology of melanoma cells grown in EGF(+)bFGF(+) medium. Fast growing populations DMBC12 and DMBC19 formed cell aggregates, whereas slow growing DMBC17 population formed more dense melanospheres. Scale bar, 100 μm. (B) qRT-PCR was used to assess the expression of MITF and MITF-dependent genes in melanoma cultures in EGF(+)bFGF(+) medium. Data are presented as the means ± SD (n = 4). A fold change more than 1,000 is shown as 1,000. * <i>p</i><0.05; *** <i>p</i><0.001; ns, <i>p</i>>0.05. (C) qRT-PCR was used to assess the expression of pro-survival genes in melanoma cells grown in EGF(+)bFGF(+) medium. Data are presented as the means ± SD (n = 4). * <i>p</i><0.05; ** <i>p</i><0.01; ns, <i>p</i>>0.05.</p

Immediate response to changes in microenvironment involves alterations in MITF level and ERK-1/2 activity.

<p>(A) Level of MITF mRNA was analyzed at indicated time points during early adaptation period of melanoma cells to serum-containing medium. Data are presented as the means ± SD from three independent experiments. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. (B) Immunodetection of phosphorylated ERK-1/2^{Thr202/Tyr204} (p-ERK-1/2) was used a readout of their activity (n = 3). Localization and molecular weight (MW) of marker bands are indicated. ERK-1/2 was used as a loading control. Quantification of p-ERK-1/2 level is shown above the blots.</p

MCL-1, BCL-X_L and MITF Are Diversely Employed in Adaptive Response of Melanoma Cells to Changes in Microenvironment

<div><p>Melanoma cells can switch their phenotypes in response to microenvironmental insults. Heterogeneous melanoma populations characterized by long-term growth and a high self-renewal capacity can be obtained <i>in vitro</i> in EGF(+)bFGF(+) medium whilst invasive potential of melanoma cells is increased in serum-containing cultures. In the present study, we have shown that originally these patient-derived melanoma populations exhibit variable expression of pro-survival genes from the BCL-2 family and inhibitors of apoptosis (IAPs), and differ in the baseline MCL-1 transcript stability as well. While being transferred to serum-containing medium, melanoma cells are well protected from death. Immediate adaptive response of melanoma cells selectively involves a temporary MCL-1 increase, both at mRNA and protein levels, and BCL-X_L can complement MCL-1, especially in MITF_low populations. Thus, the extent of MCL-1 and BCL-XL contributions seems to be cell context-dependent. An increase in MCL-1 level results from a transiently enhanced stability of its transcript, but not from altered protein turnover. Inhibition of MCL-1 preceding transfer to serum-containing medium caused the induction of cell death in a subset of melanoma cells, which confirms the involvement of MCL-1 in melanoma cell survival during the rapid alteration of growth conditions. Additionally, immediate response to serum involves the transient increase in MITF expression and inhibition of ERK-1/2 activity. Uncovering the mechanisms of adaptive response to rapid changes in microenvironment may extend our knowledge on melanoma biology, especially at the stage of dissemination.</p></div

MCL-1 is involved in melanoma cell survival during adaptation to serum-containing medium.

<p>DMBC12 cells with silenced expression of MCL-1 or BCL-X_L (0h) were exposed to serum-containing medium (FBS(+)), or in parallel they were transferred to fresh EGF(+)bFGF(+) medium. (A) Cell lysates were prepared at indicated time points and MCL-1 and BCL-X_L proteins were immunoblotted (n = 3). Localization and molecular weight (MW) of marker bands are indicated. β-actin was used as a loading control. (B) Cells were stained with Annexin V/propidium iodide (PI) before (0h) and 25 h after medium exchange, and analyzed by flow cytometry. The percentage of Annexin V-positive cells is shown. Representative counter plots for melanoma cells after transfection with either control or MCL-1 siRNA are shown. They were acquired 25 h after transfected cell were transferred to either fresh EGF(+)bFGF(+) medium or serum-containing medium (FBS(+)). Data are presented as the means ± SD from three independent experiments. (C) Quantitative analysis of the frequency of melanoma cells stained with acridine orange/ethidium bromide before (0 h) and 25 h after medium exchange. The percentage of apoptotic/necrotic cells is shown. Representative microphotographs are shown below. Data are presented as the means ± SD from three independent experiments. * <i>p</i><0.05.</p