Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

Lipase-mediated detoxification of host-derived antimicrobial fatty acids by Staphylococcus aureus.

Long-chain fatty acids with antimicrobial properties are abundant on the skin and mucosal surfaces, where they are essential to restrict the proliferation of opportunistic pathogens such as Staphylococcus aureus. These antimicrobial fatty acids (AFAs) elicit bacterial adaptation strategies, which have yet to be fully elucidated. Characterizing the pervasive mechanisms used by S. aureus to resist AFAs could open new avenues to prevent pathogen colonization. Here, we identify the S. aureus lipase Lip2 as a novel resistance factor against AFAs. Lip2 detoxifies AFAs via esterification with cholesterol. This is reminiscent of the activity of the fatty acid-modifying enzyme (FAME), whose identity has remained elusive for over three decades. In vitro, Lip2-dependent AFA-detoxification was apparent during planktonic growth and biofilm formation. Our genomic analysis revealed that prophage-mediated inactivation of Lip2 was rare in blood, nose, and skin strains, suggesting a particularly important role of Lip2 for host - microbe interactions. In a mouse model of S. aureus skin colonization, bacteria were protected from sapienic acid (a human-specific AFA) in a cholesterol- and lipase-dependent manner. These results suggest Lip2 is the long-sought FAME that exquisitely manipulates environmental lipids to promote bacterial growth in otherwise inhospitable niches.

PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Introduction: Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. Methods: In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. Results: We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Discussion: Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Dormancy of cutaneous melanoma.

Many cancer-related deaths including melanoma result from metastases that develop months or years after the initial cancer therapy. Even the most effective drugs and immune therapies rarely eradicate all tumor cells. Instead, they strongly reduce cancer burden, permitting dormant cancer cells to persist in niches, where they establish a cellular homeostasis with their host without causing clinical symptoms. Dormant cancers respond poorly to most drugs and therapies since they do not proliferate and hide in niches. It therefore remains a major challenge to develop novel therapies for dormant cancers. In this review we focus on the mechanisms regulating the initiation of cutaneous melanoma dormancy as well as those which are involved in reawakening of dormant cutaneous melanoma cells. In recent years the role of neutrophils and niche components in reawakening of melanoma cells came into focus and indicate possible future therapeutic applications. Sophisticated in vitro and in vivo melanoma dormancy models are needed to make progress in this field and are discussed.

Histological and functional characterization of 3D human skin models mimicking the inflammatory skin diseases psoriasis and atopic dermatitis.

Three-dimensional (3D) human skin equivalents have emerged as valuable tools in skin research, replacing animal experimentation and precluding the need for patient biopsies. In this study, we advanced 3D skin equivalents to model the inflammatory skin diseases atopic dermatitis and psoriasis by cytokine stimulation, and were successful in integrating TH1 T cells into skin models to develop an immunocompetent 3D psoriasis model. We performed in-depth histological and functional characterization of 3D skin equivalents and validated them in terms of tissue architecture, pathological changes, expression of antimicrobial peptides and Staphylococcus aureus colonization using 3D reconstruction by multiphoton microscopy and phenotyping by highly multiplexed 'co-detection by indexing' (CODEX) microscopy. We show that our skin equivalents have a structural architecture with a well-developed dermis and epidermis, thus resembling human skin. In addition, the skin models of atopic dermatitis and psoriasis show several phenotypic features of inflammatory skin disease, including disturbed epidermal differentiation and alterations in the expression of epidermal barrier genes and antimicrobial peptides, and can be reliably used to test novel treatment strategies. Therefore, these 3D equivalents will be a valuable tool in experimental dermatological research.

PARP Inhibitors Effectively Reduce MAPK Inhibitor Resistant Melanoma Cell Growth and Synergize with MAPK Inhibitors through a Synthetic Lethal Interaction In Vitro and In Vivo.

The efficacy of targeting the MAPK signaling pathway in patients with melanoma is limited by the rapid development of resistance mechanisms that result in disease relapse. In this article, we focus on targeting the DNA repair pathway as an antimelanoma therapy, especially in MAPK inhibitor resistant melanoma cells using PARP inhibitors. We found that MAPK inhibitor resistant melanoma cells are particularly sensitive to PARP inhibitor treatment due to a lower basal expression of the DNA damage sensor ataxia-telangiectasia mutated (ATM). As a consequence, MAPK inhibitor resistant melanoma cells have decreased homologous recombination repair activity leading to a reduced repair of double-strand breaks caused by the PARP inhibitors. We validated the clinical relevance of our findings by ATM expression analysis in biopsies from patients with melanoma before and after development of resistance to MAPK inhibitors. Furthermore, we show that inhibition of the MAPK pathway induces a homologous recombination repair deficient phenotype in melanoma cells irrespective of their MAPK inhibitor sensitivity status. MAPK inhibition results in a synthetic lethal interaction of a combinatorial treatment with PARP inhibitors, which significantly reduces melanoma cell growth in vitro and in vivo. In conclusion, this study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression. Significance: We show that MAPK inhibitor resistant melanoma cells exhibit low ATM expression increasing their sensitivity toward PARP inhibitors and that a combination of MAPK/PARP inhibitors act synthetically lethal in melanoma cells. Our study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression, which could serve as a novel biomarker for treatment response.

Neutrophil extracellular traps enhance S. aureus skin colonization by oxidative stress induction and downregulation of epidermal barrier genes.

Staphylococcus aureus is the most common cause of bacterial skin infections in humans, including patients with atopic dermatitis (AD). Polymorphonuclear neutrophils (PMNs) are the first cells to infiltrate an infection site, where they usually provide an effective first line of defense, including neutrophil extracellular trap (NET) formation. Here, we show that infiltrating PMNs in inflamed human and mouse skin enhance S. aureus skin colonization and persistence. Mechanistically, we demonstrate that a crosstalk between keratinocytes and PMNs results in enhanced NET formation upon S. aureus infection, which in turn induces oxidative stress and expression of danger-associated molecular patterns such as high-mobility-group-protein B1 (HMGB1) in keratinocytes. In turn, HMGB1 enhances S. aureus skin colonization and persistence by promoting skin barrier dysfunctions by the downregulation of epidermal barrier genes. Using patient material, we show that patients with AD exhibit enhanced presence of PMNs, NETs, and HMGB1 in the skin, demonstrating the clinical relevance of our finding.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion.

BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

IL-17 Signaling in Keratinocytes Orchestrates the Defense against Staphylococcus aureus Skin Infection.

Keratinocytes (KCs) form the outer epithelial barrier of the body, protecting against invading pathogens. Mice lacking the IL-17RA or both IL-17A and IL-17F develop spontaneous Staphylococcusaureus skin infections. We found a marked expansion of T17 cells, comprised of RORγt-expressing γδ T cells and T helper 17 cells in the skin-draining lymph nodes of these mice. Contradictory to previous suggestions, this expansion was not a result of a direct negative feedback loop because we found no expansion of T17 cells in mice lacking IL-17 signaling specifically in T cells. Instead, we found that the T17 expansion depended on the microbiota and was observed only when KCs were deficient for IL-17RA signaling. Indeed, mice that lack IL-17RA only in KCs showed an increased susceptibility to experimental epicutaneous infection with S. aureus together with an accumulation of IL-17A-producing γδ T cells. We conclude that deficiency of IL-17RA on KCs leads to microbiota dysbiosis in the skin, which triggers the expansion of IL-17A-producing T cells. Our data show that KCs are the primary target cells of IL-17A and IL-17F, coordinating the defense against microbial invaders in the skin.

The epidermal lipid barrier in microbiome-skin interaction.

The corneocyte layers forming the upper surface of mammalian skin are embedded in a lamellar-membrane matrix which repels harmful molecules while retaining solutes from subcutaneous tissues. Only certain bacterial and fungal taxa colonize skin surfaces. They have ways to use epidermal lipids as nutrients while resisting antimicrobial fatty acids. Skin microorganisms release lipophilic microbe-associated molecular pattern (MAMP) molecules which are largely retained by the epidermal lipid barrier. Skin barrier defects, as in atopic dermatitis, impair lamellar-membrane integrity, resulting in altered skin microbiomes, which then include the pathogen Staphylococcus aureus. The resulting increased penetration of MAMPs and toxins promotes skin inflammation. Elucidating how microorganisms manipulate the epidermal lipid barrier will be key for better ways of preventing inflammatory skin disorders.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Enhanced expression of p21 promotes sensitivity of melanoma cells towards targeted therapies.

Metastatic melanoma patients benefit from the approved targeted BRAF inhibitor (BRAFi) therapy. Despite the great progress in the therapeutic approach to combat metastatic melanoma, fast emerging drug resistance in patients limits its long-term efficacy. In this study, we aimed to unravel the role of the p53 target gene CDKN1A/p21 in the response of melanoma cells towards BRAFi. We show that p53 activation increases BRAFi sensitivity in a synergistic manner exclusively in cells with a high expression of CDKN1A/p21. In a similar way, high expression of p21 was associated with a better response towards the mouse double minute 2 inhibitor (MDM2i) compared to those with low p21 expression. Indeed, p21 knockdown decreased the sensitivity towards both targeted therapies. The results indicate that the sensitivity of melanoma cells towards targeted therapies (BRAFi and MDM2i) is dependent on the p21 protein level in the cells. In addition to that, we found that p53 negatively regulates p73 expression; however, p73 seems not to have an influence on p53 expression. These findings offer new potential strategies for the treatment improvement of melanoma patients with high basal p21 levels with BRAFi by increasing treatment efficacy using combination therapies with p53 activating substances, which are able to further increase p21 expression levels. Furthermore, the data suggest that the expression and induction level of p21 could be used as a predictive biomarker in melanoma patients to forecast the outcome of a treatment with p53 activating substances and BRAFi. All in all, this manuscript shows the distinct role of p53 family members and its impact on melanoma therapy. In future, individualized treatment regimens based on p21 basal and induction levels could help melanoma patients with limited treatment options.

Bacterial membrane vesicles shape Staphylococcus aureus skin colonization and induction of innate immune responses.

Staphylococcus aureus colonization is abundant on the skin of atopic dermatitis (AD) patients where it contributes to skin inflammation. S. aureus produces virulence factors that distinguish it from commensal skin bacteria such as S. epidermidis and S. lugdunensis. However, it has remained unclear, which of these virulence factors have the strongest impact on AD. Membrane vesicles (MVs) are released by pathogenic bacteria and might play an essential role in the long-distance delivery of bacterial effectors such as virulence factors. We show that MVs are also released by skin commensals in a similar quantity and membrane lipid amount as those from pathogenic S. aureus. Interestingly, MVs from skin commensals can protect against S. aureus skin colonization by conditioning human skin for enhanced defence. In contrast, MVs released by S. aureus are able to induce CXCL8 and TNF-α in primary human keratinocytes, recruit neutrophils and induce neutrophil extracellular traps, which enhance S. aureus skin colonization. CXCL8 induction is TLR2- and NFkB-dependent and the induction level correlates with the membrane lipid and protein A content of the MVs. Interestingly, MVs of S. aureus strains from the lesional skin of AD patients show an enhanced membrane lipid and protein A content compared to the strains from the non-lesional sites and have an enhanced proinflammatory potential. Our data underline the complex interplay in host- and bacterial derived factors in S. aureus skin colonization and the important role of bacterial derived MVs and their membrane lipid and protein A content in skin inflammatory disorders.

Targeting the Y-box Binding Protein-1 Axis to Overcome Radiochemotherapy Resistance in Solid Tumors.

Multifunctional Y-box binding protein-1 (YB-1) is highly expressed in different human solid tumors and is involved in various cellular processes. DNA damage is the major mechanism by which radiochemotherapy (RCT) induces cell death. On induction of DNA damage, a multicomponent signal transduction network, known as the DNA damage response, is activated to induce cell cycle arrest and initiate DNA repair, which protects cells against damage. YB-1 regulates nearly all cancer hallmarks described to date by participating in DNA damage response, gene transcription, mRNA splicing, translation, and tumor stemness. YB-1 lacks kinase activity, and p90 ribosomal S6 kinase and AKT are the key kinases within the RAS/mitogen-activated protein kinase and phosphoinositide 3-kinase pathways that directly activate YB-1. Thus, the molecular targeting of ribosomal S6 kinase and AKT is thought to be the most effective strategy for blocking the cellular function of YB-1 in human solid tumors. In this review, after describing the prosurvival effect of YB-1 with a focus on DNA damage repair and cancer cell stemness, clinical evidence will be provided indicating an inverse correlation between YB-1 expression and the treatment outcome of solid tumors after RCT. In the interest of being concise, YB-1 signaling cascades will be briefly discussed and the current literature on YB-1 posttranslational modifications will be summarized. Finally, the current status of targeting the YB-1 axis, especially in combination with RCT, will be highlighted.

Beneficial effects of coagulase-negative Staphylococci on Staphylococcus aureus skin colonization.

Our skin is constantly exposed to a large number of pathogens while at the same time undergoing selective colonization by commensal microorganisms such as coagulase-negative Staphylococci. Staphylococcus aureus, however, is a facultative pathogen that is usually absent from healthy skin but frequently colonizes the inflamed skin of atopic dermatitis patients, where it further promotes inflammation. Enhanced S. aureus skin colonization was shown to correlate with a loss of microbiome diversity indicating a role for skin commensals to shape pathogen colonization. Together, keratinocytes and immune cells in the skin need to discriminate commensals from pathogens and orchestrate subsequent immune reactions in response to colonizing microbes. However, the mechanisms how individual commensals cooperate with keratinocytes and the immune system of the skin to prevent pathogen colonization are barely understood. In this review, we discuss the current knowledge on the functional effects of coagulase-negative staphylococci, the most frequently isolated skin commensals, on S. aureus skin colonization.

Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers.

The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

Tumour Progression Stage-Dependent Secretion of YB-1 Stimulates Melanoma Cell Migration and Invasion.

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition.

Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.