AKT/PKB (Protein Kinase B) are central proteins mediating signals from receptor tyrosine kinases and phosphatidylinositol 3-kinase. AKT kinases are involved in a number of important cellular ...processes including cell proliferation and survival, cell size in response to nutrient availability, tumor invasion/metastasis, and angiogenesis.Various components of the AKT signaling pathway are encoded by tumor suppressor genes and oncogenes whose loss or activation, respectively, plays an important role in tumorigenesis. The growing body of evidence connecting deregulated AKT signaling with sporadic human cancers and inherited cancer predisposition syndromes is discussed. We also highlight new findings regarding the involvement of activating mutations of AKT1, AKT2, and AKT3 in somatic overgrowth disorders: Proteus syndrome, hypoglycemia with hypertrophy, and hemimegalencephaly, respectively. In addition, we review recent literature documenting the various ways the AKT signaling pathway is activated in human cancers and consequences for molecularly targeted therapies.
Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing ...factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.
Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why ...mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1(+/-) knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1(+/-) mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1(+/-) mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1(+/-) mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1(+/-) mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1(+/-) mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1(+/-) mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1(+/-) mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure.
Purpose: mTOR (mammalian target of rapamycin) plays a central role in regulating cell growth and cell cycle progression and is regarded
as a promising therapeutic target. We examined whether mTOR ...inhibition by RAD001 (everolimus) is therapeutically efficacious
in the treatment of ovarian cancer as a single agent and in combination with cisplatin.
Experimental Design: Using four human ovarian cancer cell lines, we determined the effect of RAD001 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, Western blot, and apoptosis assays. We evaluated the association between phospho-AKT/mTOR activity and RAD001 sensitivity.
We also determined the effect of RAD001 on tumor growth and malignancy using mice inoculated with human ovarian cancer cells.
Results: RAD001 markedly inhibited cell proliferation of human ovarian carcinoma cells with high AKT activity (OVCAR10 and SKOV-3),
but the effect was minimal in cells with low AKT activity (OVCAR4 and OVCAR5). Sensitivity to RAD001 was independent of p53
expression. RAD001 inhibited the phosphorylation of downstream 4E-BP1 and p70S6 kinase and attenuated the expression of Myc.
RAD001 also attenuated the expression of HIF-1α and vascular endothelial growth factor, important factors in angiogenesis
and tumor invasiveness. RAD001 enhanced cisplatin-induced apoptosis in cells with high AKT/mTOR activity, with minimal effect
in cells with low AKT-mTOR activity. Mouse xenografts of SKOV-3 cells revealed that RAD001 inhibits tumor growth, angiogenesis,
and i.p. dissemination and ascites production and prolongs survival. Moreover, treatment with RAD001 significantly enhanced
the therapeutic efficacy of cisplatin in vivo .
Conclusion: These results indicate that RAD001 could have therapeutic efficacy in human ovarian cancers with hyperactivated AKT/mTOR
signaling.
The distal-less homeobox gene (dlx) 5 encodes a transcription factor that controls jaw formation and appendage differentiation during embryonic development. We had previously found that Dlx5 is ...overexpressed in an Akt2 transgenic model of T-cell lymphoma. To investigate if DLX5 is involved in human cancer, we screened its expression in the NCI 60 cancer cell line panel. DLX5 was frequently upregulated in cell lines derived from several tumor types, including ovarian cancer. We next validated its upregulation in primary ovarian cancer specimens. Stable knockdown of DLX5 by lentivirus-mediated transduction of short hairpin RNA (shRNA) resulted in reduced proliferation of ovarian cancer cells due to inhibition of cell cycle progression in connection with the downregulation of cyclins A, B1, D1, D2, and E, and decreased phosphorylation of AKT. Cell proliferation resumed following introduction of a DLX5 cDNA harboring wobbled mutations at the shRNA-targeting sites. Cell proliferation was also rescued by transduction of a constitutively active form of AKT. Intriguingly, downregulation of IRS-2 and MET contributed to the suppression of AKT signaling. Moreover, DLX5 was found to directly bind to the IRS-2 promoter and augmented its transcription. Knockdown of DLX5 in xenografts of human ovarian cancer cells resulted in markedly diminished tumor size. In addition, DLX5 was found to cooperate with HRAS in the transformation of human ovarian surface epithelial cells. Together, these data suggest that DLX5 plays a significant role in the pathogenesis of some ovarian cancers.
Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby ...suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action.
In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 microg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines.
Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling.
These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental
retardation, autism, and tumors in the brain, retina, kidney, heart, and ...skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found
that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation
of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating
domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with
B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with
and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf
inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this,
inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which
is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent
function of tuberin.
Like cancer generally, malignant mesothelioma (MM) is a genetic disease at the cellular level. DNA copy number analysis of mesothelioma specimens has revealed a number of recurrent sites of ...chromosomal loss, including 3p21.1, 9p21.3, and 22q12.2. The key inactivated driver genes located at 9p21.1 and 22q12.2 were discovered two decades ago as being the tumor suppressor loci
CDKN2A
and
NF2
, respectively. Only relatively recently was the
BAP1
gene determined to be the driver gene at 3p21.1 that is somatically inactivated. In 2011, we reported germline mutations in
BAP1
in two families with a high incidence of mesothelioma and other cancers such as uveal melanoma (UM). As a result of a flurry of research activity over the last 5–6 years, the
BAP1
gene is now firmly linked causally to a novel tumor predisposition syndrome (TPDS) characterized by increased susceptibility to mesothelioma, UM, cutaneous melanoma (CM) and benign melanocytic tumors, as well as several other cancer types. Moreover, results from recent
in vivo
studies with genetically engineered
Bap1
-mutant mouse models and new functional studies have provided intriguing biological insights regarding
BAP1
’s role in tumorigenesis. These and other recent findings offer new possibilities for novel preventative and therapeutic strategies for MM patients.
Heritable mutations in the BAP1 tumor suppressor gene predispose individuals to mesothelioma and other cancers. However, a large-scale assessment of germline BAP1 mutation incidence and associated ...clinical features in mesothelioma patients with a family history of cancer has not been reported. Therefore, we examined the germline BAP1 mutation status of 150 mesothelioma patients with a family history of cancer, 50 asbestos-exposed control individuals with a family history of cancers other than mesothelioma, and 153 asbestos-exposed individuals without familial cancer. No BAP1 alterations were found in control cohorts, but were identified in nine of 150 mesothelioma cases (6%) with a family history of cancer. Alterations among these cases were characterized by both missense and frameshift mutations, and enzymatic activity of BAP1 missense mutants was decreased compared with wild-type BAP1. Furthermore, BAP1 mutation carriers developed mesothelioma at an earlier age that was more often peritoneal than pleural (five of nine) and exhibited improved long-term survival compared to mesothelioma patients without BAP1 mutations. Moreover, many tumors harboring BAP1 germline mutations were associated with BAP1 syndrome, including mesothelioma and ocular/cutaneous melanomas, as well as renal, breast, lung, gastric, and basal cell carcinomas. Collectively, these findings suggest that mesothelioma patients presenting with a family history of cancer should be considered for BAP1 genetic testing to identify those individuals who might benefit from further screening and routine monitoring for the purpose of early detection and intervention.
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