Persistent Parental RNAi in the Beetle Tribolium castaneum Involves Maternal Transmission of Long Double-Stranded RNA.

Parental RNA interference (pRNAi) is a powerful and widely used method for gene-specific knockdown. Yet in insects its efficacy varies between species, and how the systemic response is transmitted from mother to offspring remains elusive. Using the beetle Tribolium castaneum, an RT-qPCR strategy to distinguish the presence of double-stranded RNA (dsRNA) from endogenous mRNA is reported. It is found that injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, it is shown that strong pRNAi can persist for months before waning at strain-specific rates. In seeking the receptor proteins for cellular uptake of long dsRNA into the egg, a phylogenomics profiling approach of candidate proteins is also presented. A visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by sequence-based evidence, is demonstrated. Repeated losses of SID-1-like channel proteins in the arthropods, including wholesale loss in the Heteroptera (true bugs), which are nonetheless highly sensitive to pRNAi, are thereby documented. Overall, practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission for long-term, systemic knockdown are elucidated.

Persistent Parental RNAi in the Beetle Tribolium castaneum Involves Maternal Transmission of Long Double-Stranded RNA (Advanced Genetics 3/03).

dsRNA Uptake In article 2100064 by Kristen A. Panfilio and co-workers, the cuticle exoskeleton of flour beetle larvae reveals normal anatomy (above: head-to-tail in blue-to-red) and long-term parental RNAi knockdown (below), here showing a mirror-image duplication of the abdomen (red termini to yellow center). Strong knockdown can persist for months despite transmission of full-length double-stranded RNA (dsRNA) from the mother into the egg, depleting maternal dsRNA levels.

Efficacy of Dual Inhibition of Glycolysis and Glutaminolysis for Therapy of Renal Lesions in Tsc2+/- Mice.

Tuberous sclerosis is caused by mutations in the TSC1 or TSC2 gene and characterized by development of tumors in multiple organs including the kidneys. TSC-associated tumors exhibit somatic loss of the second allele of the TSC genes, leading to aberrant activation of the mechanistic target of rapamycin (mTOR) signaling pathway. Activation of mTOR complex 1 (mTORC1) causes addiction to glucose and glutamine in Tsc1-/-or Tsc2-/- mouse embryonic fibroblasts (MEFs). Blocking of glutamine anaplerosis in combination with glycolytic inhibition causes significant cell death in Tsc2-/- but not Tsc2+/+ MEFs. In this study, we tested efficacy of dual inhibition of glycolysis with 3-BrPA and glutaminolysis with CB-839 for renal tumors in Tsc2+/- mice. Following 2 months of treatment of Tsc2+/- mice from the age of 12 months, combination of 3-BrPA and CB-839 significantly reduced overall size and cellular areas of all renal lesions (cystic/papillary adenomas and solid carcinomas), but neither alone did. Combination of 3-BrPA and CB-839 inhibited mTORC1 and the proliferation of tumor cells but did not increase apoptosis. However, combination of 3-BrPA and CB-839 was not as efficacious as rapamycin alone or rapamycin in combination with either 3-BrPA or CB-839 for renal lesions of Tsc2+/- mice. Consistently, rapamycin alone or rapamycin in combination with either 3-BrPA or CB-839 had stronger inhibitory effects on mTORC1 and proliferation of tumor cells than combination of 3-BrPA and CB-839. We conclude that combination of 3-BRPA and CB-839 may not offer a better therapeutic strategy than rapamycin for TSC-associated tumors.

Allosteric and ATP-Competitive Inhibitors of mTOR Effectively Suppress Tumor Progression-Associated Epithelial-Mesenchymal Transition in the Kidneys of Tsc2+/- Mice.

In tuberous sclerosis (TSC)-associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2+/- mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2+/- mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.

Assessment of Response of Kidney Tumors to Rapamycin and Atorvastatin in Tsc1+/- Mice.

Atorvastatin is widely used to lower blood cholesterol and to reduce risk of cardiovascular disease-associated complications. Epidemiological investigations and preclinical studies suggest that statins such as atorvastatin have antitumor activity for various types of cancer. Tuberous sclerosis (TSC) is a tumor syndrome caused by TSC1 or TSC2 mutations that lead to aberrant activation of mTOR and tumor formation in multiple organs. Previous studies have demonstrated that atorvastatin selectively suppressed growth and proliferation of mouse Tsc2 null embryonic fibroblasts through inhibition of mTOR. However, atorvastatin alone did not reduce tumor burden in the liver and kidneys of Tsc2+/- mice as assessed by histological analysis, and no combination therapy of rapamycin and atorvastatin has been tried. In this study, we used T2-weighted magnetic resonance imaging to track changes in tumor number and size in the kidneys of a Tsc1+/- mouse model and to assess the efficacy of rapamycin and atorvastatin alone and as a combination therapy. We found that rapamycin alone or rapamycin combined with atorvastatin significantly reduced tumor burden, while atorvastatin alone did not. Combined therapy with rapamycin and atorvastatin appeared to be more effective for treating renal tumors than rapamycin alone, but the difference was not statistically significant. We conclude that combined therapy with rapamycin and atorvastatin is unlikely to provide additional benefit over rapamycin as a single agent in the treatment of Tsc-associated renal tumors.

The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis.

Tuberous sclerosis (TSC) is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR. Tumour responses in TSC patients to rapamycin, an allosteric inhibitor of mTOR, or its analogs are partial and reversible probably due to feedback activation of Akt. In this study, we examined the efficacy of GSK2126458, an ATP-competitive dual inhibitor of PI3K/mTOR, in comparison to rapamycin for treatment of renal tumours in genetically engineered Tsc2+/- mice. We found that both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 also significantly reduced the number and size of all lesions (cystic, papillary and solid) although to a lesser extent compared to rapamycin. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Furthermore, GSK2126458 and rapamycin suppressed proliferation of tumour cells. Importantly, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are therefore needed to test whether rapamycin in combination with GSK2126458 could promote apoptosis and thus improve therapy of TSC-associated renal tumours.

Combination of Everolimus with Sorafenib for Solid Renal Tumors in Tsc2+/- Mice Is Superior to Everolimus Alone.

Tuberous sclerosis (TSC) is an inherited tumor syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR and development of tumors in multiple organs including the kidneys. The mTOR inhibitors rapamycin and everolimus (rapalogs) have demonstrated clinical efficacy in treating TSC-associated tumors including renal angiomyolipomas. However, tumor responses are usually only partial, and regrowth occurs after drug withdrawal. TSC-associated tumors are highly vascular, and TSC patients with renal angiomyolipomas have elevated levels of circulating vascular endothelial growth factor (VEGF) A and VEGFD. Sorafenib inhibits multiple kinases including VEGF receptors and has been used to treat metastatic epithelioid angiomyolipoma in one case, but formal trials have not been undertaken. In this study, we investigated tumor angiogenesis and the therapeutic efficacy of everolimus in combination with sorafenib for renal tumors in Tsc2+/- mice. We found that these tumors exhibited remarkably variable angiogenesis despite consistent aberrant activation of mTOR and increased expression of HIF1α and VEGFA. Treatment of 11-month-old Tsc2+/- mice for 2 months with a combination of everolimus and sorafenib significantly reduced the number and size of solid renal tumors, whereas everolimus or sorafenib alone did not. These results suggest that inhibition of mTOR and multiple kinases including VEGF receptors using combination therapy could hold promise for the treatment of TSC-associated tumors that have responded inadequately to a rapalog alone.