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Carcinogenesis is a multistep process. Besides somatic mutations in tumor cells, stroma-associated immunity is a major regulator of tumor growth. Tumor cells produce and secrete diverse mediators to create a local microenvironment that supports their own survival and growth. It is becoming apparent that iron acquisition, storage, and release in tumor cells is different from healthy counterparts. It is also appreciated that macrophages in the tumor microenvironment acquire a tumor-supportive, anti-inflammatory phenotype that promotes tumor cell proliferation, angiogenesis, and metastasis. Apparently, this behavior is attributed, at least in part, to the ability of macrophages to support tumor cells with iron. Polarization of macrophages by apoptotic tumor cells shifts the profile of genes involved in iron metabolism from an iron sequestering to an iron-release phenotype. Iron release from macrophages is supposed to be facilitated by ferroportin. However, lipid mediators such as sphingosine-1-phosphate, released form apoptotic tumor cells, upregulate lipocalin-2 (Lcn-2) in macrophages. This protein is known to bind siderophore-complexed iron and thus, may participate in iron transport in the tumor microenvironment. We describe how macrophages handle iron in the tumor microenvironment, discuss the relevance of an iron-release macrophage phenotype for tumor progression, and propose a new role for Lcn-2 in tumor-associated macrophages.
Macrophages supply iron to the breast tumor microenvironment by enforced secretion of lipocalin-2 (Lcn-2)-bound iron as well as the increased expression of the iron exporter ferroportin (FPN). We aimed at identifying the contribution of each pathway in supplying iron for the growing tumor, thereby fostering tumor progression. Analyzing the expression profiles of Lcn-2 and FPN using the spontaneous polyoma-middle-T oncogene (PyMT) breast cancer model as well as mining publicly available TCGA (The Cancer Genome Atlas) and GEO Series(GSE) datasets from the Gene Expression Omnibus database (GEO), we found no association between tumor parameters and Lcn-2 or FPN. However, stromal/macrophage-expression of Lcn-2 correlated with tumor onset, lung metastases, and recurrence, whereas FPN did not. While the total iron amount in wildtype and Lcn-2−/− PyMT tumors showed no difference, we observed that tumor-associated macrophages from Lcn-2−/− compared to wildtype tumors stored more iron. In contrast, Lcn-2−/− tumor cells accumulated less iron than their wildtype counterparts, translating into a low migratory and proliferative capacity of Lcn-2−/− tumor cells in a 3D tumor spheroid model in vitro. Our data suggest a pivotal role of Lcn-2 in tumor iron-management, affecting tumor growth. This study underscores the role of iron for tumor progression and the need for a better understanding of iron-targeted therapy approaches.
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis.
Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone.
Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.
Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.
Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.
Background: Glucose metabolism in the tumor-microenvironment is a fundamental hallmark for tumor growth and intervention therein remains an attractive option for anti-tumor therapy. Whether tumor-derived factors such as microRNAs (miRs) regulate glucose metabolism in stromal cells, especially in tumor-associated macrophages (TAMs), to hijack them for trophic support, remains elusive.
Methods: Ago-RIP-Seq identified macrophage lactate dehydrogenase B (LDHB) as a target of tumor-derived miR-375 in both 2D/3D cocultures and in murine TAMs from a xenograft mouse model. The prognostic value was analyzed by ISH and multiplex IHC of breast cancer patient tissues. Functional consequences of the miR-375-LDHB axis in TAMs were investigated upon mimic/antagomir treatment by live metabolic flux assays, GC/MS, qPCR, Western blot, lentiviral knockdown and FACS. The therapeutic potential of a combinatorial miR-375-decoy/simvastatin treatment was validated by live cell imaging.
Results: Macrophage LDHB decreased in murine and human breast carcinoma. LDHB downregulation increase aerobic glycolysis and lactagenesis in TAMs in response to tumor-derived miR-375. Lactagenesis reduced fatty acid synthesis but activated SREBP2, which enhanced cholesterol biosynthesis in macrophages. LDHB downregulation skewed TAMs to function as a lactate and sterol/oxysterol source for the proliferation of tumor cells. Restoring of LDHB expression potentiated inhibitory effects of simvastatin on tumor cell proliferation.
Conclusion: Our findings identified a crucial role of LDHB in macrophages and established tumor-derived miR-375 as a novel regulator of macrophage metabolism in breast cancer, which might pave the way for strategies of combinatorial cancer cell/stroma cell interventions.
Loss of HIF-1α in macrophages attenuates AhR/ARNT-mediated tumorigenesis in a PAH-driven tumor model
(2016)
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.
Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade.
Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1.
Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism.
Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.
Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.
In solid tumors, tumor‐associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia‐inducible factors (HIFs). While HIF‐1α is ubiquitously expressed, HIF‐2α appears tissue‐specific with consequences of HIF‐2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF‐2α knockout (HIF‐2αLysM−/−) compared with wildtype (wt) mice. In an RNA‐sequencing approach of FACS sorted wt and HIF‐2α LysM−/− TAMs, serine protease inhibitor, Kunitz type‐1 ( Spint1) emerged as a promising candidate for HIF‐2α‐dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF‐2α‐deficient bone marrow–derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF‐2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro‐HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor‐suppressive function of HIF‐2α in TAMs in breast tumor development.
Background: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells.
Aims: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets.
Methods: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used.
Results: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented.
Conclusions: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.
Macrophage S1PR1 signaling alters angiogenesis and lymphangiogenesis during skin inflammation
(2019)
The bioactive lipid sphingosine-1-phosphate (S1P), along with its receptors, modulates lymphocyte trafficking and immune responses to regulate skin inflammation. Macrophages are important in the pathogenesis of psoriasiform skin inflammation and express various S1P receptors. How they respond to S1P in skin inflammation remains unknown. We show that myeloid specific S1P receptor 1 (S1PR1) deletion enhances early inflammation in a mouse model of imiquimod-induced psoriasis, without altering the immune cell infiltrate. Mechanistically, myeloid S1PR1 deletion altered the formation of IL-1β, VEGF-A, and VEGF-C, and their receptors’ expression in psoriatic skin, which subsequently lead to reciprocal regulation of neoangiogenesis and neolymphangiogenesis. Experimental findings were corroborated in human clinical datasets and in knockout macrophages in vitro. Increased blood vessel but reduced lymph vessel density may explain the exacerbated inflammatory phenotype in conditional knockout mice. These findings assign a novel role to macrophage S1PR1 and provide a rationale for therapeutically targeting local S1P during skin inflammation.
While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
MicroRNAs (miRs) significantly contribute to the regulation of gene expression, by virtue of their ability to interact with a broad, yet specific set of target genes. MiRs are produced and released by almost every cell type and play an important role in horizontal gene regulation in the tumor microenvironment (TME). In the TME, both tumor and stroma cells cross-communicate via diverse factors including miRs, which are taking central stage as a therapeutic target of anti-tumor therapy. One of the immune escape strategies adopted by tumor cells is to release miRs as a Trojan horse to hijack circulating or tumor-localized monocytes/macrophages to tune them for pro-tumoral functions. On the other hand, macrophage-derived miRs exert anti-tumor functions. The transfer of miRs from host to recipient cells depends on the supramolecular structure and composition of miR carriers, which determine the distinct uptake mechanism by recipient cells. In this review, we provide a recent update on the miR-mediated crosstalk between tumor cells and macrophages and their mode of uptake in the TME.
Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME.
Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.
Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.