Conditional disruption of rictor demonstrates a direct requirement for mTORC2 in skin tumor development and continued growth of established tumors.

Activation of signaling dependent on the mammalian target of rapamycin (mTOR) has been demonstrated in a variety of human malignancies, and our previous work suggests that mTOR complex (mTORC) 1 and mTORC2 may play unique roles in skin tumorigenesis. The purpose of these studies was to investigate the function of mTORC2-dependent pathways in skin tumor development and the maintenance of established tumors. Using mice that allow spatial and temporal control of mTORC2 in epidermis by conditional knockout of its essential component Rictor, we studied the effect of mTORC2 loss on both epidermal proliferation and chemical carcinogenesis. The results demonstrate that mTORC2 is dispensable for both normal epidermal proliferation and the hyperproliferative response to treatment with tetradecanoyl phorbol acetate (TPA). In contrast, deletion of epidermal Rictor prior to initiation in DMBA/TPA chemical carcinogenesis was sufficient to dramatically delay tumor development and resulted in reduced tumor number and size compared with control groups. Silencing of Rictor expression in tumor-bearing animals triggered regression of established tumors and increased caspase-3 cleavage without changes in proliferation. In vitro experiments demonstrate an increased sensitivity to caspase-dependent apoptosis in the absence of rictor, which is dependent on mTORC2 signaling. These studies demonstrate that mTORC2 activation is essential for keratinocyte survival, and suggest that inhibition of mTORC2 has value in chemoprevention by eliminating carcinogen-damaged cells during the early stages of tumorigenesis, and in therapy of existing tumors by restricting critical pro-survival pathways.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells.

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3'UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3'UTR that may act as regulatory sequences. Analysis of ODC 3'UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells.

Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2 +/- 9.6%) and c-raf-1 mRNA levels (86.2 +/- 3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-XL), cell adhesion (paxillin, plectin, Rho GDIalpha, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.

Inhibition of mTOR suppresses UVB-induced keratinocyte proliferation and survival.

UV radiation is the major risk factor for developing skin cancer, the most prevalent cancer worldwide. Several studies indicate that mTOR signaling is activated by UVB and may play an important role in skin tumorigenesis. mTOR exists in two functionally and compositionally distinct protein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2). The purpose of these studies was to investigate the roles of the two mTOR complexes in UVB-mediated proliferation and apoptosis in the skin. We used rapamycin, a pharmacologic inhibitor of mTORC1, and an inducible mTOR-deficient (K5-CreER(T2);mTOR(fl/fl)) mouse model that allows epidermal-specific disruption of mTOR following topical treatment with 4-hydroxytamoxifen (4OHT). Rapamycin blocked UVB-induced phosphorylation of S6K, the downstream target of mTORC1, and significantly reduced UVB-stimulated epidermal proliferation and cell-cycle progression, but had no effect on cell death. In contrast, mTOR deletion, which attenuated UVB-induced phosphorylation of both S6K and the mTORC2 target AKT(Ser473), significantly increased apoptosis both in vivo and in keratinocyte cultures, in addition to reducing hyperproliferation following UVB irradiation. The role of mTORC2 in UVB-induced prosurvival signaling was verified in Rictor(-/-) mouse embryo fibroblasts, which lack functional mTORC2 and were more sensitive to UVB-induced apoptosis than controls. These studies show that mTORC1 and mTORC2 play unique but complementary roles in controlling proliferation and apoptosis in the skin. Our findings underscore the importance of both mTOR complexes in mediating UVB-induced signaling in keratinocytes and provide new insight into the pathogenesis of skin cancer.