Transformation Of Fibroblasts Research Articles

MiR-155 and let-7a potentiate auto-inflammatory and tumor-like proliferative dysregulation in post-infectious Lyme arthritis synovial tissue

Abstract Post-infectious Lyme arthritis (LA) is a model for testing the causal relationship between infection and autoimmunity. The initial trigger for LA, Borrelia burgdorferi infection, is known with certainty, but it is unclear why some patients have persistent synovitis after completion of antibiotic therapy. To elucidate the inflammatory nature of post-infectious LA, whole transcriptome sequencing was performed on synovial tissue from 14 patients with post-infectious LA and compared with that from patients with other forms of inflammatory arthritis or degenerative arthritis. LA patient synovial tissue had a highly inflammatory expression signature, similar to patients with other immune disorders including Type IV hypersensitivity and autoimmune diseases. This profile included upregulation of IFN-responsive genes and genes associated with MHC Class II antigen processing and presentation, T cell activation, and cell-mediated cytotoxicity; and down-regulation of genes involved in tissue repair and cell metabolism. Fibroblasts isolated from synovial tissue and primary fibroblasts in vitro expressed markers of inflammation and active proliferation, consistent with the tumor-like proliferative nature of the synovial lesion. miR-155, a proinflammatory and oncogenic microRNA, was highly expressed and positively correlated with markers of inflammation and Class II antigen presentation. Expression of tumor suppressor let-7a was downregulated, negatively correlated with markers of inflammation, and positively correlated with markers of appropriate wound repair. We postulate that these two microRNAs potentiate epigenetic transformation of synovial fibroblasts into an auto-inflammatory and proliferative phenotype.

Th1 Effector T cells induce TGFβ mediated Cardiac Fibrosis in Pressure Overload induced Heart Failure by adhering to Alpha 4 Integrin and mediating Fibroblast Transformation

Cardiac fibrosis (CF) is a key factor contributing to the pathogenesis of the deadly syndrome of heart failure (HF). Recent emerging data demonstrate a role for T cells in pro‐fibrotic cardiac repair and healing post‐ischemia. However, very little is known about how T cells directly impact cardiac fibroblasts (CFB) to promote CF and cardiac dysfunction in non‐ischemic HF. Recently, we reported that T cells infiltrated the left ventricle (LV) of the heart in patients with end‐stage non‐ischemic HF, and also in the mouse model of non‐ischemic HF induced by thoracic aortic constriction (TAC). Interestingly, mice deficient in T cells (TCRα−/− mice) are protected from cardiac fibrosis (CF) and cardiac dysfunction. Thus, we hypothesize that T cells activated in such context orchestrate the activation and differentiation of fibroblasts to myofibroblasts. To test this hypothesis, we first co‐cultured adult CFB with T cells isolated from mice 4 weeks post TAC or Sham surgery. TAC but not Sham T cells firmly adhered to CFB and mediated myofibroblasts transformation as demonstrated by increased α‐SMA fluorescent expression. Co‐culture of various CD4 T cell subsets with CFB demonstrated that Th1 effector T cell subset, in contrast to naïve T cells, adhered directly to CFB through alpha 4 integrin and mediated myofibroblasts transformation in a TGFβ dependent manner. Moreover, adoptive transfer (AT) of effector Th1 cells, but not naïve T cells into TCRα−/− mice at 48 hours and 2 weeks post TAC surgery exclusively induced perivascular fibrosis that co‐localized with T cell and leukocyte infiltration and TGFβ expression. AT of Th1 cells also induced LV contractile and systolic dysfunction and a shift in the fetal gene expression of the myosin heavy chain α toβ which occurs in pathological cardiac remodeling. Taken together, these findings identify a novel role for Th1 cells in cardiac fibrosis and function and may provide innovative insights in the treatment of chronic non‐ischemic HF.Support or Funding InformationNIH HL094706

Piperine Attenuates Pathological Cardiac Fibrosis Via PPAR-γ/AKT Pathways

Mitogen-activated protein kinases (MAPKs) and AMP­activated protein kinase α (AMPKα) play critical roles in the process of cardiac hypertrophy. Previous studies have demonstrated that piperine activates AMPKα and reduces the phosphorylation of extracellular signal-regulated kinase (ERK). However, the effect of piperine on cardiac hypertrophy remains completely unknown. Here, we show that piperine-treated mice had similar hypertrophic responses as mice treated with vehicle but exhibited significantly attenuated cardiac fibrosis after pressure overload or isoprenaline (ISO) injection. Piperine inhibited the transformation of cardiac fibroblasts to myofibroblasts induced by transforming growth factor-β (TGF-β) or angiotensin II (Ang II) in vitro. This anti-fibrotic effect was independent of the AMPKα and MAPK pathway. Piperine blocked activation of protein kinase B (AKT) and, downstream, glycogen synthase kinase 3β (GSK3β). The overexpression of constitutively active AKT or the knockdown of GSK3β completely abolished the piperine-mediated protection of cardiac fibroblasts. The cardioprotective effects of piperine were blocked in mice with constitutively active AKT. Pretreatment with GW9662, a specific inhibitor of peroxisome proliferator activated receptor-γ (PPAR-γ), reversed the effect elicited by piperine in vitro. In conclusion, piperine attenuated cardiac fibrosis via the activation of PPAR-γ and the resultant inhibition of AKT/GSK3β.

Myocardial fibrosis in congenital and pediatric heart disease.

Cardiac fibrosis is a common phenomenon in different types of heart diseases, such as ischemic heart disease, inherited cardiomyopathy mutations, diabetes, and ageing and is associated with morbidity and mortality. Increased accumulation of extracellular matrix (ECM) that impacts cardiac function, is the underlying cause of fibrotic heart disease. There are four different types of cardiac fibrosis, including, reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis and endomyocardial fibrosis. They are involved in the activation and transformation of cardiac fibroblasts to myofibroblasts, which participate in ECM production and fibrotic process and several inflammatory pathways. Besides the ECM proteins, myofibroblasts also express smooth muscle α-actin, SM22 and caldesmon and other markers related to fibrotic process. Most commonly employed techniques to assess myocardial fibrosis include stress echocardiography, cardiac magnetic resonance imaging and positron emission tomography. Because of the involvement of renin-angiotensin-II-aldosterone system, transforming growth factor-β signaling and activin-linked kinase 5 in the mechanisms of cardiac fibrosis, these pathways and the involved proteins are useful as therapeutic targets. However, because of the importance of these pathways in many other physiological functions, their therapeutic targeting needs to be approached with caution.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.

Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro.

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

Intensification and redistribution of protrusive activity is a feature of tumor transformation and is associated with an increase of the invasive potential of cells

The abilities of tumor cells to invade and metastasize are frequent causes of death of cancer patients. Studying the mechanisms of cell motility alterations and acquisition of enhanced metastatic potential as the result of transformation is an important aspect in current cell biology. The initial and determinant step of cell motility is the formation of active cell edge with protrusions based on the Arp2/3-dependent actin polymerization. We used three different cell systems as examples of different models of tumor transformation to study the alteration and redistribution of protrusive activity caused by transformation in fibroblasts. We analyzed relationships between detected alterations and the acquisition of increased invasive potential by cells. Active edge of untransformed fibroblasts occupies about 50% of the cell perimeter and is concentrated at the cell front. There are well pronounced stable regions at the lateral cell edges. Tumor transformation causes redistribution of protrusive activity of fibroblasts irrespective of their origin and the nature of transforming agents. The length of active edges significantly increases, up to 92% of the total perimeter in fibrosarcoma cells of tumor origin. These cells have practically no stable edges. The intensity of protrusive activity of transformed cells is also increased. Single transformed cells show a decrease in the directionality and rate of migration on 2D substrate without special stimulation. Instead, they gain the capacity to migrate in 3D and to invade matrigel. These abilities increase in parallel with the intensification of edge activity. We showed that invasive abilities are not associated with the activation of matrix metalloproteinases in the studied cell systems. Our data demonstrate that the increase of length of active edge could be considered as an additional feature of cell transformation together with the reduction of stress fiber and focal adhesions and that the excessive protrusive activity results in the development of explorative migration of tumor cells.

Multiple Myeloma-Derived Exosomes Regulate the Functions of Mesenchymal Stem Cells Partially via Modulating miR-21 and miR-146a.

Exosomes derived from cancer cells can affect various functions of mesenchymal stem cells (MSCs) via conveying microRNAs (miRs). miR-21 and miR-146a have been demonstrated to regulate MSC proliferation and transformation. Interleukin-6 (IL-6) secreted from transformed MSCs in turn favors the survival of multiple myeloma (MM) cells. However, the effects of MM exosomes on MSC functions remain largely unclear. In this study, we investigated the effects of OPM2 (a MM cell line) exosomes (OPM2-exo) on regulating the proliferation, cancer-associated fibroblast (CAF) transformation, and IL-6 secretion of MSCs and determined the role of miR-21 and miR-146a in these effects. We found that OPM2-exo harbored high levels of miR-21 and miR-146a and that OPM2-exo coculture significantly increased MSC proliferation with upregulation of miR-21 and miR-146a. Moreover, OPM2-exo induced CAF transformation of MSCs, which was evidenced by increased fibroblast-activated protein (FAP), α-smooth muscle actin (α-SMA), and stromal-derived factor 1 (SDF-1) expressions and IL-6 secretion. Inhibition of miR-21 or miR-146a reduced these effects of OPM2-exo on MSCs. In conclusion, MM could promote the proliferation, CAF transformation, and IL-6 secretion of MSCs partially through regulating miR21 and miR146a.

Compositions of Anticancer Drug with Micellar Nanocarriers and Their Cytotoxicity

Asymmetric diblock (DBC) and triblock (TBC) copolymers contained biocompatible chemically complementary polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) or its monomethyl ether (MEPEO-b-PAAm), and also partially hydrolyzed triblock copolymer derivative (TBChydr) were used to create micelles of a special type. The micelles obtained are characterized by small CMCs and large values of the Gibbs micellization energy, thus indicating a high stability of DBC, TBC and TBChydr micelles in aqueous solutions and the capabilities of their use to encapsulate and deliver poorly soluble and/or toxic drugs in living organism. Morphological features and size of DBC and TBC micelles were determined by TEM. The electron images demonstrated spherical micelles of a polymolecular type, monomolecular type and separate micelle aggregates. TBC and TBChydr micelles were used to examine in vitro anticancer activity of their compositions with doxorubicin (Dox). The created micelle systems showed the enhanced cytotoxicity as compared to individual Dox against murine leukemia cells of L1210 line, murine transformed fibroblasts of L929 line and human T-leukemia cells of Jurkat line and allow to achieve a high efficacy at low Dox concentrations (0,1÷3 µg·cm-3) that opens the great prospects for essential decrease in drug dose at chemotherapy.

BCL6 Is Critical to Overcome Oncogene-Induced Senescence in RAS-Mediated B Cell Transformation

Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 is a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and modulates drug-resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., Nature 2011). BCL6 was shown to be a critical factor that bypasses p53-dependent senescence and thereby enables RAS-driven transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev. 2002). Given that ~50% of pediatric ALL cases carry genetic lesions that lead to hyperactivated RAS-ERK signaling (Zhang et la., Blood 2012), we examined the role of BCL6 in RAS-driven pre-B ALL and identified a novel mechanism by which RAS-ERK signaling can mediate BCL6 expression.Results: Using a doxycycline-inducible TetOn- NRASG12D vector system, we found that inducible activation of RAS-ERK signaling strongly upregulated BCL6 expression at both the mRNA (~350-fold) and protein (~50-fold) levels in murine pre-B cells. Increases in BCL6 expression were abrogated upon treatment with a MEK inhibitor (PD325901). In addition, Cre-mediated deletion of Mapk1 suppressed upregulation of BCL6 expression upon imatinib treatment in BCR-ABL1-driven pre-B ALL cells. These findings suggested that elevated expression of BCL6 is a consequence of ERK activation. Previously, we demonstrated that BCL6 expression is negatively regulated by STAT5 in BCR-ABL1 pre-B ALL (Duy et al., Nature 2011). Interestingly, oncogenic NRASG12D inhibited phosphorylation of STAT5-Y694 by activating the inhibitory protein tyrosine phosphatase Ptpn6. Cre-mediated deletion of Ptpn6 induced STAT5 activity. Furthermore, loss of Ptpn6 function abrogated upregulation of BCL6 expression induced by imatinib in BCR-ABL1 pre-B ALL. Taken together, RAS-ERK signaling induces BCL6 expression by suppressing STAT5 activity. To directly test the role of BCL6 in RAS-transformed pre-B ALL, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. Inducible deletion of Bcl6 in NRASG12D-transformed pre-B ALL cells led to rapid depletion from the cell culture and reduced colony forming ability in vitro. These findings suggested that BCL6 is required for maintenance of fully established RAS-transformed ALL. Notably, we found that initiation of NRASG12D-driven leukemia in vivo depends on BCL6 as NRASG12D ALL failed to give rise to leukemia in the absence of Bcl6 in transplant recipient mice. Studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed increased BCL6 expression in KRASG12V relapsed ALL cells. In addition, selective sensitivity to PD325901 and a retro inverso BCL6 peptide inhibitor (RI-BPI) was observed in KRASG12V relapsed ALL cells. Finally, RI-BPI prolonged overall survival of recipient mice transplanted with KRASG12V relapsed ALL cells in vivo.Conclusions: In summary, we demonstrated a novel mechanism by which oncogenic RAS signaling induces expression of BCL6, and showed that BCL6 is critical for RAS-driven transformation in pre-B ALL. Importantly, ALL clones often acquire drug resistance and activating mutations in the RAS pathway (Bhojwani and Pui, Lancet Oncol. 2013). Our findings suggest that pharmacological inhibition of BCL6 may provide a novel therapeutic avenue to overcome drug-resistance and prevent leukemia relapse after initial remission in RAS-driven ALL. DisclosuresMelnick:Janssen: Research Funding.

Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.

Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis.

BackgroundThe abnormal epithelial-mesenchymal restorative capacity in idiopathic pulmonary fibrosis (IPF) has been recently associated with an accelerated aging process as a key point for the altered wound healing. The advanced glycation end-products (AGEs) are the consequence of non-enzymatic reactions between lipid and protein with several oxidants in the aging process. The receptor for AGEs (RAGEs) has been implicated in the lung fibrotic process and the alveolar homeostasis. However, this AGE-RAGE aging pathway has been under-explored in IPF.MethodsLung samples from 16 IPF and 9 control patients were obtained through surgical lung biopsy. Differences in AGEs and RAGE expression between both groups were evaluated by RT-PCR, Western blot and immunohistochemistry. The effect of AGEs on cell viability of primary lung fibrotic fibroblasts and alveolar epithelial cells was assessed. Cell transformation of fibrotic fibroblasts cultured into glycated matrices was evaluated in different experimental conditions.ResultsOur study demonstrates an increase of AGEs together with a decrease of RAGEs in IPF lungs, compared with control samples. Two specific AGEs involved in aging, pentosidine and Nε-Carboxymethyl lysine, were significantly increased in IPF samples. The immunohistochemistry identified higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs) surrounding fibroblast foci in fibrotic lungs. On the other hand, RAGE location was present at the cell membrane of AECs in control lungs, while it was almost missing in pulmonary fibrotic tissue. In addition, in vitro cultures showed that the effect of AGEs on cell viability was different for AECs and fibrotic fibroblasts. AGEs decreased cell viability in AECs, even at low concentration, while fibroblast viability was less affected. Furthermore, fibroblast to myofibroblast transformation could be enhanced by ECM glycation.ConclusionsAll of these findings suggest a possible role of the increased ratio AGEs-RAGEs in IPF, which could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.

Smad Nuclear Interacting Protein 1 Acts as a Protective Regulator of Pressure Overload-Induced Pathological Cardiac Hypertrophy.

BackgroundSmad nuclear interacting protein 1 (SNIP1) plays a critical role in cell proliferation, transformation of embryonic fibroblasts, and immune regulation. However, the role of SNIP1 in cardiac hypertrophy remains unclear.Methods and ResultsHere we examined the role of SNIP1 in pressure overload–induced cardiac hypertrophy and its mechanisms. Our results demonstrated that SNIP1 expression was downregulated in human dilated cardiomyopathic hearts, aortic banding‐induced mice hearts, and angiotensin II–treated cardiomyocytes. Accordingly, SNIP1 deficiency significantly exacerbated aortic banding–induced cardiac hypertrophy, fibrosis, and contractile dysfunction, whereas cardiac‐specific overexpression of SNIP1 markedly recovered pressure overload–induced cardiac hypertrophy and fibrosis. Besides that, SNIP1 protected neonatal rat cardiomyocytes against angiotensin II–induced hypertrophy in vitro. Moreover, we identified that SNIP1 suppressed nuclear factor‐κB signaling during pathological cardiac hypertrophy, and inhibition of nuclear factor‐κB signaling by a cardiac‐specific conditional inhibitor of κBS 32A/S36A transgene blocked these adverse effects of SNIP1 deficiency on hearts.ConclusionsTogether, our findings demonstrated that SNIP1 had protective effects in pressure overload–induced pathological cardiac hypertrophy via inhibition of nuclear factor‐κB signaling. Thus, SNIP1 may be a novel approach for the treatment of heart failure.

Protective role of ACE2-Ang-(1-7)-Mas in myocardial fibrosis by downregulating KCa3.1 channel via ERK1/2 pathway.

The intermediate-conductance Ca2+-activated K+ (KCa3.1) channel plays a vital role in myocardial fibrosis induced by angiotensin (Ang) II. However, as the antagonists of Ang II, the effect of angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas axis on KCa3.1 channel during myocardial fibrosis remains unknown. This study was designed to explore the function of KCa3.1 channel in the cardioprotective role of ACE2-Ang-(1-7)-Mas. Wild-type (WT) mice, hACE2 transgenic mice (Tg), and ACE2 deficiency mice (ACE2-/-) were administrated with Ang II by osmotic mini-pumps. As the activator of ACE2, diminazene aceturate (DIZE) inhibited increase of blood pressure, collagen deposition, and KCa3.1 protein expression in myocardium of WT mice induced by Ang II. In Tg and ACE2-/- mice, besides the elevation of blood pressure, Ang II induced transformation of cardiac fibroblast into myofibroblast and resulted in augmentation of hydroxyproline concentration and collagen deposition, as well as KCa3.1 protein expression, but the changes in ACE2-/- mice were more obvious than those in Tg mice. Mas antagonist A779 reduced blood pressure, myocardium fibrosis, and myocardium KCa3.1 protein expression by Ang II in Tg mice, but activation of KCa3.1 with SKA-31 in Tg mice promoted the pro-fibrogenic effects of Ang II. Respectively, in ACE2-/- mice, TRAM-34, the KCa3.1 blocker, and Ang-(1-7) inhibited increase of blood pressure, collagen deposition, and KCa3.1 protein expression by Ang II. Moreover, DIZE and Ang-(1-7) depressed p-ERK1/2/t-ERK increases by Ang II in WT mice, and after blockage of ERK1/2 pathway with PD98059, the KCa3.1 protein expression was reduced in WT mice. In conclusion, the present study demonstrates that ACE2-Ang-(1-7)-Mas protects the myocardium from hypertension-induced injury, which is related to its inhibiting effect on KCa3.1 channels through ERK1/2 pathway. Our results reveal that KCa3.1 channel is likely to be a critical target on the ACE2-Ang-(1-7)-Mas axis for its protective role in myocardial fibrosis and changes of KCa3.1 induced by homeostasis of ACE-Ang II-AT1 axis and ACE2-Ang-(1-7)-Mas axis may be a new therapeutic target in myocardial fibrosis.

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.

S18 family of mitochondrial ribosomal proteins: evolutionary history and Gly132 polymorphism in colon carcinoma.

S18 family of mitochondrial ribosomal proteins (MRPS18, S18) consists of three members, S18-1 to −3. Earlier, we found that overexpression of S18-2 protein resulted in immortalization and eventual transformation of primary rat fibroblasts. The S18-1 and −3 have not exhibited such abilities. To understand the differences in protein properties, the evolutionary history of S18 family was analyzed. The S18-3, followed by S18-1 and S18-2 emerged as a result of ancient gene duplication in the root of eukaryotic species tree, followed by two metazoan-specific gene duplications. However, the most conserved metazoan S18 homolog is the S18-1; it shares the most sequence similarity with S18 proteins of bacteria and of other eukaryotic clades. Evolutionarily conserved residues of S18 proteins were analyzed in various cancers. S18-2 is mutated at a higher rate, compared with S18-1 and −3 proteins. Moreover, the evolutionarily conserved residue, Gly132 of S18-2, shows genetic polymorphism in colon adenocarcinomas that was confirmed by direct DNA sequencing.Concluding, S18 family represents the yet unexplored important mitochondrial ribosomal proteins.

Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay

High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a −40%, a “normal” and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development.

Abstract 5068: Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression

Abstract The tumor microenvironment (TME) is a heterogeneous region comprised of tumor cells, stromal cells, and secreted factors that make the environment favorable for cancer formation and progression. An important step in the shift to a pro-cancerous environment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are a critical cell type to understand. They are present in a majority of solid tumors and can have a direct effect to enhance adjacent tumor cell motility via their innate ability to secret cytokines, chemokine and growth factors into the TME. We sought to understand how CAF-derived chemokines impact breast tumor cell motility through modification of the F-actin cytoskeleton. We collected cultured media (CM) from WS19T fibroblasts, a patient-derived breast tumor CAF cell line. CAF-CM dramatically enhanced wound-closure in MDA-MB-231 monolayers, relative to normal fibroblast cultured media. The chemokine CXCL12 (SDF1 alpha) has been identified as a potential secreted CAF-directed signal driving tumor cell migration. To determine if CXCL12 was the soluble factor in CAF-CM promoting tumor cell migration, MDA-MB-231 cells were preincubated with AMD3100, an inhibitor of the CXCL12 receptor, CXCR4 prior to CAF-CM application and wounding. AMD3100 effectively blocked CAF-CM-induced MDA-MB-231 migration. CXCL12 incubation with MDA-MB-231 cells equally promoted MDA-MB-231 wound closure, supporting the notion that CXCL12 is secreted by CAFs to promote MDA-MB-231 motility. We previously showed a link between CXCL12 directed CXCR4 signaling and a critical regulator of the dynamic F-actin cytoskeleton, mammalian Diaphanous-related formin (mDia2). This formin regulates the assembly and bundling of unbranched actin filaments and plays a role in tumor cell migration and invasion programs. Western blotting CAF-CM-treated MDA-MB-231 cells revealed a near complete loss of mDia2 protein. mDia1 and ROCK expression were unaffected with CAF-CM treatment. These data were consistent with treatment with a functional mDia FH2-domain inhibitor SMIFH2, which likewise suppresses mDia2 protein levels in MDA-MB-231 cells. It remains unclear if CAF-derived CXCL12 first suppresses mDia2 FH2 activity, leading to loss of mDia2 protein, thus promoting tumor cell motility. Current experiments are designed to address this gap in the knowledge, potentially indicating a role for CAF-derived soluble factors in modulating mDia2 protein function and expression in migrating tumor cells. Citation Format: Kaitlyn Dvorak, Kathryn M. Eisenmann. Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5068.

Protective role for miR-9-5p in the fibrogenic transformation of human dermal fibroblasts.

BackgroundExcessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis.ResultsmiR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model.ConclusionsmiR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-016-0044-2) contains supplementary material, which is available to authorized users.

Transforming growth factor-β-dependent Wnt secretion controls myofibroblast formation and myocardial fibrosis progression in experimental autoimmune myocarditis.

Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-β (TGF-β) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/β-catenin pathway in response to TGF-β signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-β-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/β-catenin-mediated transcription with ICG-001 or genetic loss of β-catenin also prevented TGF-β-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-β-activated kinase 1 (TAK1) pathway completely abrogated TGF-β-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-β, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/β-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-β-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.