ABSTRACT
OBJECTIVE: We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. DESIGN: We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. RESULTS: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. CONCLUSIONS: Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/genetics , Adult , Aged , Animals , Antibiotics, Antineoplastic/pharmacology , Axin Protein/genetics , Carcinoma, Hepatocellular/chemistry , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , DNA Mutational Analysis , DNA-Binding Proteins , Female , Humans , Liver Neoplasms/chemistry , Liver Neoplasms/metabolism , Male , Mice , Middle Aged , Mutation Rate , Neoplasm Transplantation , Nuclear Proteins/genetics , Signal Transduction , Sirolimus/pharmacology , Transcription Factors/genetics , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Stem Cell Assay , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/analysis , Young Adult , beta Catenin/geneticsABSTRACT
Spinal muscular atrophy (SMA) is the most common genetic motoneuron degenerative disorder, but the mechanism(s) of motoneuron degeneration is unclear. We previously generated SMA model mice, which genotypically and phenotypically mimicked human SMA patients, by a combination of knockout and transgenic techniques. Here, we used these SMA model mice to decipher the apoptotic mechanism(s) involved in SMA motoneuron degeneration. We found a significant increase in proapoptotic Bax expression in the spinal cords of SMA mice in comparison with their wild-type littermates. After crossing SMA mice with Bax knockout mice, we produced in vivo evidence indicating that Bax protein plays an important role in the degeneration of SMA spinal motoneurons. Progeny Bax-deficient SMA mice showed milder disease severity, longer life spans, and significant increases in spinal motoneuron densities compared to SMA littermates with wild-type Bax genes. Our results strongly suggest that suppression of Bax-involved apoptosis has the potential for amelioration of SMA.
