The miR-34 family and its clinical significance in ovarian cancer.

The tumor suppressor miR-34 family is transcriptionally induced by p53. Clinical significance of the various miR-34 family members has not been studied in ovarian cancer. In 228 ovarian cancers and in 19 non-neoplastic fallopian tube samples we analysed miR-34 a/b/c expression in relation to clinicopathological characteristics and clinical outcome. We found significantly lower levels of miR-34 a/b/c in ovarian cancers as compared to control-tissues (P=0.002, P<0.001, P<0.001, respectively). Expression of miR-34 b/c revealed an inverse correlation with BRCA1/2 mRNA-expression (BRCA1: miR34 b/c P=0.002 each; BRCA2: miR-34 b/c P<0.001 each), the same was true for miR-34a and BRCA2 mRNA-expression (P<0.001). The miR-34 family expression was found to be significantly lower in type 2 in comparison to type 1 cancers (P<0.001) and in TP53-mutated compared with TP53-wild-type ovarian cancers (P<0.001, P=0.002, P=0.004, respectively). When low grade serous ovarian cancers were compared with high grade serous cancers the respective miR-34 a/b/c expression was 2.6-, 40.8- and 32.3-fold higher. The expression of each of the miR-34 family members was revealed to be of independent prognostic relevance regarding progression free survival (PFS); miR-34a: HR 0.6, P=0.033; miR-34b: HR 0.2, P=0.001 and miR-34c: HR 0.3, P=0.002, respectively). For overall survival (OS) independency of the prognostic value was confined to miR-34b (HR 0.4, P=0.016) and miR-34c (HR 0.6, P=0.049). The independency of the prognostic value of our identified thresholds was confirmed for PFS for miR-34c in a publicly available dataset (NCBI Gene Expression Omnibus GSE73582). Our findings suggest that downregulation of miR-34 family is a crucial part in ovarian cancer development. Low miR-34 levels are linked to a worse overall survival and progression free survival and may indicate a more aggressive disease.

Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions.

Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.

BRCA1 and BRCA2 mRNA-expression prove to be of clinical impact in ovarian cancer.

BACKGROUND: Mutations in BRCA1 and BRCA2 are associated with better survival in ovarian cancer (OC) patients due to a better response to platinum-based chemotherapy. However, the impact of the BRCA1/2 mRNA-expression is not well characterized in OC. PATIENTS AND METHODS: We investigated BRCA1/2 mRNA-expression in 12 non-neoplastic fallopian tubes and 201 epithelial OCs in relation to their clinical characteristics. RESULTS: We found higher BRCA1/2 mRNA-expression in OCs compared to controls (P = 0.011, P < 0.001, respectively). BRCA1 mutated OCs exhibited lower BRCA1 (P = 0.014) but higher BRCA2 mRNA-expression (P = 0.001). Low BRCA1-expression was associated with favorable overall survival (OS) (P = 0.012) and low BRCA2-expression with better progression-free survival (PFS) and OS (P = 0.004, P = 0.001, respectively). A subgroup-analysis showed that this effect was confined only to the BRCA1-wildtype cancers. Cox-regression confirmed the prognostic significance of BRCA1-expression for OS (P = 0.028). Independency of the prognostic value of BRCA2-expression for PFS (P = 0.045) and OS (P = 0.015) was restricted to high-grade serous OCs. Fully platinum-sensitivity was characterized by lower BRCA1/2 mRNA-expression in BRCA1-wildtype cancers in comparison to platinum-refractory OC. CONCLUSION: Our findings may reflect higher platinum-sensitivity due to reduced capacity of DNA damage repair in tissues with low BRCA1/2-expression. In this context, especially in BRCA-wildtype cancers both parameters could also be potential predictors for PARP-sensitivity.

BRCA1/2 and TP53 mutation status associates with PD-1 and PD-L1 expression in ovarian cancer.

Checkpoint molecules such as programmed cell death protein-1 (PD-1) and its ligand PD-L1 are critically required for tumor immune escape. The objective of this study was to investigate tumoral PD-1 and PD-L1 mRNA-expression in a cohort of ovarian cancer (OC) patients in relation to tumor mutations. We analyzed mRNA expression of PD-1, PD-L1 and IFNG by quantitative real-time PCR in tissue of 170 patients with low grade-serous (LGSOC), high-grade serous (HGSOC), endometrioid and clear cell OC compared to 28 non-diseased tissues (ovaries and fallopian tubes) in relation to tumor protein 53 (TP53) and breast cancer gene 1/2 (BRCA1/2) mutation status. TP53-mutated OC strongly expressed PD-L1 compared to TP53 wild-type OC (p = 0.028) and BRCA1/2-mutated OC increasingly expressed PD-1 (p = 0.024) and PD-L1 (p = 0.012) compared to BRCA1/2 wild-type OC. For the first time in human, we noted a strong correlation between tumoral IFNG and PD-1 or PD-L1 mRNA-expression, respectively (p < 0.001). OC tissue increasingly expressed PD-1 compared to healthy controls (vs. ovaries: p < 0.001; vs. tubes: p = 0.018). PD-1 and PD-L1 mRNA-expression increased with higher tumor grade (p = 0.008 and p = 0.027, respectively) and younger age (< median age, p = 0.001). Finally, in the major subgroup of our cohort, FIGO stage III/IV HGSOC, high PD-1 and PD-L1 mRNA-expression was associated with reduced progression-free (p = 0.024) and overall survival (p = 0.049) but only in the univariate analysis. Our study suggests that in OC PD-1/PD-L1 mRNA-expression is controlled by IFNγ and affected by TP53 and BRCA1/2 mutations. We suggest that these mutations might serve as potential predictive factors that guide anti-PD1/PD-L1 immunotherapy.

Crystal structure of the human lysosomal mTORC1 scaffold complex and its impact on signaling.

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.

Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR).

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated 'THT') for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.