Project description:While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.
Project description:Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.
Project description:Static mechanical compression is a biomechanical factor that affects the progression of melanoma cells. However, little is known about how dynamic mechanical compression affects the progression of melanoma cells. In the present study, we show that mechanical intermittent compression affects the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results suggest that intermittent compression with a cycle of 2 h on/2 h off could suppress the progression rate of melanoma cells by suppressing the elongation of F-actin filaments and mRNA expression levels related to collagen degradation. In contrast, intermittent compression with a cycle of 4 h on/4 h off could promote the progression rate of melanoma cells by promoting cell proliferation and mRNA expression levels related to collagen degradation. Mechanical intermittent compression could therefore affect the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results contribute to a deeper understanding of the physiological responses of melanoma cells to dynamic mechanical compression.
Project description:The development of targeted inhibitors, vemurafenib and dabrafenib, has led to improved clinical outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes causing complete tumor regression, the majority of melanomas eventually become resistant. Mitogen-activated protein kinase kinase (MEK) mutations are found in primary melanomas and frequently reported in BRAF melanomas that develop resistance to targeted therapy; however, melanoma is a molecularly heterogeneous cancer, and which mutations are drivers and which are passengers remains to be determined. In this study, we demonstrate that in BRAFV600E melanoma cell lines, activating MEK mutations drive resistance and contribute to suboptimal growth of melanoma cells following the withdrawal of BRAF inhibition. In this manner, the cells are drug-addicted, suggesting that melanoma cells evolve a 'just right' level of mitogen-activated protein kinase signaling and the additive effects of MEK and BRAF mutations are counterproductive. We also used a novel mouse model of melanoma to demonstrate that several of these MEK mutants promote the development, growth and maintenance of melanoma in vivo in the context of Cdkn2a and Pten loss. By utilizing a genetic approach to control mutant MEK expression in vivo, we were able to induce tumor regression and significantly increase survival; however, after a long latency, all tumors subsequently became resistant. These data suggest that resistance to BRAF or MEK inhibitors is probably inevitable, and novel therapeutic approaches are needed to target dormant tumors.
Project description:Autophagy is a catabolic process required for maintaining intracellular energy homeostasis. It eliminates harmful proteins and recycles functional macromolecules back into the cell via cargo breakdown. Autophagy is generally suppressed under fed conditions and induced by serum starvation; therefore, it is considered to be a nutrient-sensing mechanism. Cilia, finger-like organelles harboring multiple receptors along their surface, are energy-sensing structures that are also triggered by serum deprivation. Herein, we verified the effect of autophagy alterations on cilia assembly and the specific underlying mechanisms. Autophagy flux altered either by drugs or autophagy-targeting siRNAs strongly inhibited ciliogenesis, and this inhibition was affected by p62, an autophagy regulator, via Pten/Dvl2/AurKA signaling.
Project description:Vitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro- or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.
Project description:Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. To build a model of human melanoma, we generated mice with conditional melanocyte-specific expression of BRaf(V600E). Upon induction of BRaf(V600E) expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15-20 months. By contrast, expression of BRaf(V600E) combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma, indicating the presence of long-lived melanoma-initiating cells in this system. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanoma's cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.
Project description:Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. Here, we investigate this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. We find that the impact of altered autophagy depends on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhanced Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy.
Project description:A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU(+) cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.
Project description:KRAS mutations are found in ?90% of human pancreatic ductal adenocarcinomas (PDAC). However, mice genetically engineered to express Kras(G12D) from its endogenous locus develop PDACs only after a prolonged latency, indicating that other genetic events or pathway alterations are necessary for PDAC progression. The PTEN-controlled phosphatidylinositol 3-kinase (PI3K)/AKT signaling axis is dysregulated in later stages of PDAC. To better elucidate the role of PTEN/PI3K/AKT signaling in Kras(G12D)-induced PDAC development, we crossed Pten conditional knockout mice (Pten(lox/lox)) to mice with conditional activation of Kras(G12D). The resulting compound heterozygous mutant mice showed significantly accelerated development of acinar-to-ductal metaplasia (ADM), malignant pancreatic intraepithelial neoplasia (mPanIN), and PDAC within a year. Moreover, all mice with Kras(G12D) activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age. Our data support a dosage-dependent role for PTEN, and the resulting dysregulation of the PI3K/AKT signaling axis, in both PDAC initiation and progression, and shed additional light on the signaling mechanisms that lead to the development of ADM and subsequent mPanIN and pancreatic cancer.