BRAF(V600) inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF(V600) mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.1114sciescopu

Targeting Myc-driven tumours - BETing on ATR

Cancer arises from loss of function of tumour suppressors and/or gain of function mutations in proto-oncogenes that disrupt the delicate balance required for homeostatic cell division, resulting in uncontrolled cell proliferation. Oncogenic transformation of multifaceted proto-oncogene - transcription factor - MYC can give rise to cancers and it is found to be deregulated in more than 70% of the tumours. Targeting MYC directly or identifying the Achille’s heel of MYC-driven tumours is thus a promising therapeutic approach to treat these tumours. This thesis investigates and demonstrates novel therapeutic approaches against MYC-driven tumours. In the first publication (Bhadury et al, 2014), we characterize a novel and orally bio-available BET bromodomain inhibitor (BETi) RVX2135. We also identified BET bromodomain proteins as a valuable therapeutic target against MYC driven tumours in vitro and in vivo. Gene expression profiling to identify these transcriptional changes enabled us to identify subset of genes that are commonly altered by both BETi and HDACi. This study also demonstrates that HDACi and BETi can synergize to hinder Myc-induced lymphoma progression. The second publication (Muralidharan et al, 2016) in this thesis investigates the role of BET proteins in regulating cell cycle and replication. BETi disable the entry of cells into S-phase of cell-cycle, hamper DNA synthesis and cause DNA damage. A pharmacogenetic screen identified BET inhibitors to synergize with inhibition of PI3K/mTOR family of proteins, to which ATR, an upstream kinase of DDR pathway belongs. Further studies revealed that the thus identified PI3K/mTOR inhibitors indeed affect ATR-Chkl DDR pathway leading to the discovery of a strong synergy between BETi and ATRi in apoptosing Myc driven tumours in vitro, and in vivo and (by) it induces SASP and ER stress. The third study translates the above findings into the field of melanoma, a form of skin cancer. We validate the BETi-ATRi synergy in cell lines in vitro and in Patient Derived Xenografts (PDX) in vivo. Using B16F10 in vivo syngenic transplant melanoma model, we also demonstrated that this combination therapy can be safely combined with Immune Therapy, the front line treatment against melanoma in clinic today. Taken together, this thesis puts forth a multifaceted approach to treat cancer. It thoroughly describes the effects of BETi and ATRi on cancer cells and how they can be combined to enhance the therapeutic efficacy

Aberrant Fat Metabolism in Caenorhabditis elegans Mutants with Defects in the Defecation Motor Program

The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisinfamily, Ca2+-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steadystate levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status.supported by grants from Vetenskapsrådet (K2012-67X-20441-063) and Cancerfonden (12 0534).</p

<i>sv75</i> and <i>sv76</i> are alleles of <i>aex-5</i>.

<p>A. Micrographs of fixed young adult hermaphrodites stained with Oil Red O (top panels) or Sudan Black (bottom panels). In wild-type (N2) worms, the dyes stain the intestine and the oocytes within the gonad. In the <i>aex-5</i>(<i>sa23</i>) mutant, only the oocytes show significant staining. Scale bars are 10 μm. B. Graph showing triglyceride/phospholipid ratios determined by gas-liquid chromatography. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Fischer's test for least significant difference (σ = 0.05). C. Schematic diagram of the <i>aex-5</i> gene (at top) and protein (at bottom). White boxes and lines at top represent exons and introns respectively. The positions of the codons and amino acids affected by the <i>sv75</i> and <i>sv76</i> mutations are marked.</p

Mutations in <i>aex-1</i> and <i>aex-2</i> also cause a reduction in fat accumulation.

<p>A-C. Fixed, young adult hermaphrodite worms stained with Oil Red O. D-F. Micrographs showing part of the intestines of fixed, young adult hermaphrodite worms stained with Sudan Black. The staining in E and F is of eggs within the gonad. G-I. Fluorescence confocal micrographs of parts of young adult hermaphrodite worms fixed with isopropanol and stained with Nile Red. J. Graph showing the normalized ratios of total triglycerides to total phospholipids extracted from young adult hermaphrodite worms. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Fischer's test for least significant difference (σ = 0.05).</p

Fluorescence confocal micrographs of the intestines of young adult worms stained with Nile Red in isopropanol.

<p>The worms were subjected to RNAi by feeding after they had become adults. L4440 is the negative control in which wild-type adult worms were fed bacteria containing the plasmid vector L4440 alone. The <i>aex-5</i>(<i>RNAi</i>) worms were fed bacteria expressing double-stranded RNA from part of the <i>aex-5</i> gene.</p

<i>sv75</i> and <i>sv76</i> mutant worms have lower triglyceride/phospholipid ratios than the N2 wild-type strain.

<p>The graph shows normalised ratios of total triglycerides (TG) to total phospholipids (PL) in extracts from young adult hermaphrodites. Levels of triglycerides and phospholipids relative to C13:0 and C17:0 standards respectively were determined by gas-liquid chromatography. Three extracts were examined for each genotype. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Bonferroni test for differences between means (σ = 0.05).</p

<i>aex-5</i> mutants take up a fluorescently labelled fatty acid derivative from the intestinal lumen less efficiently than wild type.

<p>A. Confocal fluorescence micrographs of worms fed the fluorescently labelled fatty acid derivative C₁-BODIPY 500/510 C₁₂. The panels at the top show parts of the intestines of adult worms; those at the bottom show first larval stage (L1) worms. The arrows point to the intestinal lumen where most of the dye is found in the mutants. Scale bars are 20 μ m. B. Graphs showing quantification (in arbitrary units) of the uptake of the dye into the cytoplasm of intestinal cells (Materials and Methods). Error bars indicate standard errors of the means. **** indicates significant difference in the means determined by one-way ANOVA and Dunnett's multiple comparisons test (σ = 0.05).</p

Other defecation mutants also display reduced uptake of C₁-BODIPY 500/510 C₁₂.

<p>A. Confocal fluorescence micrographs of worms fed the fluorescently labelled fatty acid derivative C₁-BODIPY 500/510 C₁₂. The arrows indicate the lumens of the intestine. In all panels, the anterior part of the intestine is up and (in most) slightly to the right. Scale bars are 40 μm. B. Quantification of the uptake of the dye into the cytoplasm of intestinal cells (in arbitrary units). Error bars indicate standard errors of the means. **** indicates significant difference in the means determined by one-way ANOVA and Dunnett's multiple comparisons test (σ = 0.05).</p