Miriam Galvonas Jasiulionis

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

BackgroundAnoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.MethodsThe β1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and β1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.ResultsDifferential association among Timp1, CD63 and β1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.ConclusionsOur results show that Timp1 is assembled in a supramolecular complex containing CD63 and β1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.

Transient inflammatory response induced by apoptotic cells is an important mediator of melanoma cell engraftment and growth.

Two murine melanoma cell lines, Tm1 and Tm5, were derived from a nontumorigenic lineage of pigmented murine melanocytes, melan‐a. Both Tm1 and Tm5 are invariably tumorigenic in syngeneic mice when inoculated s.c. in doses higher than 104 cells; 103 or fewer cells rarely give rise to tumors. We demonstrate that subtumorigenic inocula of Tm1 or Tm5 cells (103) as well as of a known murine melanoma cell line (B16F10) develop as vigorously growing tumor grafts only when coinoculated with apoptotic, but not necrotic cells. The presence of apoptotic cells correlates with a transient inflammatory infiltrate, composed mainly of neutrophils and macrophages. Kinin B1 receptor–deficient mice, which have impaired transmigration of neutrophils to inflamed tissues, had significant growth inhibition of subtumorigenic doses of melanoma cells coinjected with apoptotic cells. Using the same model, tumor take in athymic mice was similar to that seen in wild‐type mice, suggesting that a T cell–dependent inflammatory response is not necessary to promote the survival and growth of subtumorigenic doses of melanoma cells. Taken together, our results describe how tumor engraftment and growth can be profoundly affected by microenvironmental alterations in response to the presence of apoptotic cells. Disrupting the delicate balance between apoptotic cells and leukocyte infiltration may provide potentially important insights for understanding and interfering with tumor cell viability during treatment with either γ‐radiation or apoptosis‐inducing drugs.

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death.

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Modification of Epigenetic Patterns in Low Birth Weight Children: Importance of Hypomethylation of the ACE Gene Promoter

There is a growing body of evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders linked to fetal programming. Angiotensin-converting enzyme (ACE) appears as one candidate gene that brings new insights into the epigenetic control and later development of diseases. In this view, we have postulated that epigenetic modifications in the ACE gene might show different interactions between birth weight (BW), blood pressure levels, plasma ACE activity and ACE I/D polymorphism. To explore this hypothesis, we performed a cross-sectional study to evaluate the DNA methylation of 3 CpG sites using pyrosequencing within the ACE gene promoter of peripheral blood leukocytes from 45 LBW children compared with 70 NBW children. Our results have revealed that LBW children have lower methylation levels (P<0.001) in parallel with a higher ACE activity (P = 0.001). Adjusting for prematurity, gender, age, body mass index, and family history of cardiovascular disease did not alter these findings. We have also performed analyses of individual CpG sites. The frequency of DNA methylation was significantly different at two CpG sites (site 1: nucleotide position +555; and site 3: nucleotide position +563). In addition, we have found a significant inverse correlation between degree of DNA methylation and both ACE activity (P<0.001) and systolic blood pressure levels (P<0.001). We also observed that the methylation level was significantly lower in LBW children who are carriers of the DD genotype compared to NBW children with DD genotype (P<0.024). In conclusion, we are able to demonstrate that the hypomethylation in the 3 CpG sites of ACE gene promoter is associated with LBW in 6 to 12 year-old children. The magnitude of these epigenetic changes appears to be clinically important, which is supported by the observation that discrete changes in DNA methylation can affect systolic blood pressure and ACE protein activity levels.

Enhanced OXPHOS, glutaminolysis and β-oxidation constitute the metastatic phenotype of melanoma cells

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Leukotriene B4 creates a favorable microenvironment for murine melanoma growth.

Chronic inflammation has long been associated with neoplastic progression. Our group had recently shown that the addition of a large number of apoptotic tumor cells to the tumor microenvironment induces a potent acute inflammatory reaction capable of promoting melanoma growth; however, primarily necrotizing cells do not cause such a reaction. Here, we show that potent inflammatory agents, such as lipopolysaccharide (LPS) and carrageenan, also promote growth of subtumorigenic doses of melanoma cells, having no effect on melanoma proliferation in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have a pivotal role in this model because caffeic acid and MK886, a FLAP (5-LOX–activating protein) inhibitor, partially hindered tumor growth induced by apoptotic cells or LPS. Other enzymes of the arachidonic acid pathway, cyclooxygenase-1 and cyclooxygenase-2, seem to have no participation in this tumor promoter effect, as the inhibitor of both enzymes (indomethacin) did not alter melanoma growth. Leukotriene B4 (LTB4), the main product of the 5-LOX pathway, was able to induce growth of subtumorigenic inocula of melanoma cells, and a LTB4 receptor antagonist inhibited acute inflammation-associated tumor growth. Addition to the tumor inflammatory microenvironment of eicosapentaenoic acid, an ω3-polyunsaturated fatty acid with anti-inflammatory properties, or leukotriene B5, an eicosapentaenoic acid–derived leukotriene, significantly inhibited tumor development. These results give new insights to the mechanisms through which inflammation may contribute to tumor progression and suggest that LOX has an important role in tumor progression associated with an inflammatory state in the presence of apoptosis, which may be a consideration for apoptosis-inducing treatments, such as chemotherapy and radiotherapy. (Mol Cancer Res 2009;7(9):1417–24)

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I–converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

Biomarkers as Key Contributors in Treating Malignant Melanoma Metastases

Melanoma is a human neurocristopathy associated with developmental defects in the neural crest-derived epidermal melanocytes. At the present time, at least three hypotheses were identified that may explain melanoma aetiology, as follows: (1) a model of linear progression from differentiated melanocytes to metastatic cancer cells (2) a model involving the appearance of melanoma stem-like cells, and (3) an epigenetic progenitor model of cancer. Treating metastatic melanoma is one of the most serious challenges in the 21st century. This is justified because of a subpopulation of cells presenting a remarkable molecular heterogeneity, which is able to explain the drug resistance and the growing mortality rates worldwide. Fortunately, there are now evidences sustaining the importance of genetic, epigenetic, and metabolomic alterations as biomarkers for classification, staging, and better management of melanoma patients. To illustrate some fascinating insights in this field, the genes BRAF V600E and CTLA4 have been recognized as bona fide targets to benefit melanoma patients. Our research attempts to carefully evaluate data from the literature in order to highlight the link between a molecular disease model and the key contribution of biomarkers in treating malignant melanoma metastases.

Ras and Rac1, Frequently Mutated in Melanomas, Are Activated by Superoxide Anion, Modulate Dnmt1 Level and Are Causally Related to Melanocyte Malignant Transformation

A melanocyte malignant transformation model was developed in our laboratory, in which different melanoma cell lines were obtained after submitting the non-tumorigenic melanocyte lineage melan-a to sequential cycles of anchorage impediment. Our group has already showed that increased superoxide level leads to global DNA hypermemethylation as well increased Dnmt1 expression few hours after melanocyte anchorage blockade. Here, we showed that Ras/Rac1/ERK signaling pathway is activated in melanocytes submitted to anchorage impediment, regulating superoxide levels, global DNA methylation, and Dnmt1 expression. Interestingly, Ras and Rac1 activation is not related to codon mutations, but instead regulated by superoxide. Moreover, the malignant transformation was drastically compromised when melan-a melanocytes were submitted to sequential cycles of anchorage blockage in the presence of a superoxide scavenger. This aberrant signaling pathway associated with a sustained stressful condition, which might be similar to conditions such as UV radiation and inflammation, seems to be an early step in malignant transformation and to contribute to an epigenetic reprogramming and the melanoma development.

Epigenetic Modulations in Activated Cells Early after HIV-1 Infection and Their Possible Functional Consequences

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.