Host cell mTORC1 is required for HCV RNA replication.

OBJECTIVE: Chronically HCV-infected orthotopic liver transplantation (OLT) recipients appear to have improved outcomes when their immunosuppressive regimen includes a mammalian target of rapamycin (mTOR) inhibitor. The mechanism underlying this observation is unknown. DESIGN: We used virological assays to investigate mTOR signalling on the HCV replication cycle. Furthermore, we analysed HCV RNA levels of 42 HCV-positive transplanted patients treated with an mTOR inhibitor as part of their immunosuppressive regimen. RESULTS: The mTOR inhibitor rapamycin was found to be a potent inhibitor for HCV RNA replication in Huh-7.5 cells as well as primary human hepatocytes. Half-maximal inhibition was observed at 0.01 µg/mL, a concentration that is in the range of serum levels seen in transplant recipients and does not affect cell proliferation. Early replication cycle steps such as cell entry and RNA translation were not affected. Knockdown of raptor, an essential component of mTORC1, but not rictor, an essential component of mTORC2, inhibited viral RNA replication. In addition, overexpression of raptor led to higher viral RNA replication, demonstrating that mTORC1, but not mTORC2, is required for HCV RNA replication. In 42 HCV-infected liver-transplanted or kidney-transplanted patients who were switched to an mTOR inhibitor, we could verify that mTOR inhibition decreased HCV RNA levels in vivo. CONCLUSIONS: Our data identify mTORC1 as a novel HCV replication factor. These findings suggest an underlying mechanism for the observed benefits of mTOR inhibition in HCV-positive OLT recipients and potentiate further investigation of mTOR-containing regimens in HCV-positive recipients of solid organ transplants.

TSH induces metallothionein 1 in thyrocytes via Gq/11- and PKC-dependent signaling.

Metallothioneins (MTs) are cytoprotective proteins acting as scavengers of toxic metal ions or reactive oxygen species. MTs are upregulated in follicular thyroid carcinoma and are regarded as a marker of thyroid stress in Graves' disease. However, the mechanism of MT regulation in thyrocytes is still elusive. In other cellular systems, cAMP-, calcium-, or protein kinase C (PKC)-dependent signaling cascades have been shown to induce MT expression. Of note, all of these three pathways are activated following the stimulation of the TSH receptor (TSHR). Thus, we hypothesized that TSH represents a key regulator of MT expression in thyrocytes. In fact, TSHR stimulation induced expression of MT isoform 1X (MT1X) in human follicular carcinoma cells. In these cells, Induction of MT1X expression critically relied on intact Gq/11 signaling of the TSHR and was blocked by chelation of intracellular calcium and inhibition of PKC. TSHR-independent stimulation of cAMP formation by treating cells with forskolin also led to an upregulation of MT1X, which was completely dependent on PKA. However, inhibition of PKA did not affect the regulation of MT1X by TSH. As in follicular thyroid carcinoma cells, TSH also induced MT1 protein in primary human thyrocytes, which was PKC dependent as well. In summary, these findings indicate that TSH stimulation induces MT1X expression via Gq/11 and PKC, whereas cAMP-PKA signaling does not play a predominant role. To date, little has been known regarding cAMP-independent effects of TSHR signaling. Our findings extend the knowledge about the PKC-mediated functions of the TSHR.

Stimulation of the chemosensory TRPA1 cation channel by volatile toxic substances promotes cell survival of small cell lung cancer cells.

TRPA1, a member of the transient receptor potential (TRP) family of cation channels, has mainly been characterized as a chemosensory protein in neuronal cells. TRPA1 is activated by toxic or irritating volatile agents like allyl isothiocyanate (AITC), tear gas, formalin, or cigarette smoke. To date, little is known about a function of TRPA1 in non-neuronal cells in the respiratory system and even less regarding a possible role in cancer biology. Here, we show that TRPA1 is expressed in a panel of human small cell lung cancer (SCLC) cell lines. Of note, TRPA1 mRNA was also significantly higher expressed in tumor samples of SCLC patients as compared to non-SCLC tumor samples or non-malignant lung tissue. Stimulation of SCLC cells with AITC led to a rise of the intracellular calcium concentration. This calcium response was inhibited by TRPA1 antagonists. Furthermore, AITC or formalin stimulated ERK1/2 in TRPA1-expressing HEK293 cells and in SCLC cells via a Src- and calcium-dependent mechanism. More importantly, TRPA1 activation in SCLC cells prevented apoptosis induced by serum starvation and thus promoted cell survival, an effect which could be blocked by inhibition of TRPA1 or ERK1/2. Vice versa, down-regulation of TRPA1 severely impaired anchorage-independent growth of SCLC cells. Since TRPA1 appears to play a pivotal role for cell survival in SCLC cells we propose that this channel could represent a promising target for therapeutic interventions. Furthermore, our data suggest that exogenous, inhalable activators of TRPA1 could be able to exert tumor promoting effects in SCLC cells.

Critical role of G(alpha)12 and G(alpha)13 for human small cell lung cancer cell proliferation in vitro and tumor growth in vivo.

PURPOSE: In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of G(q/11) and G(12/13) proteins. Although the contribution of the G(q/11)-phospholipase C-beta cascade to mitogenic effects in SCLC cells is well established, the relevance of G(12/13) signaling is still elusive. In other tumor entities, G(12/13) activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G(12/13)-dependent signaling in SCLC. EXPERIMENTAL DESIGN: We used small hairpin RNA-mediated targeting of G(alpha)(12), G(alpha)(13), or both in H69 and H209 cells and analyzed the effects of G(alpha)(12) and/or G(alpha)(13) knockdown on tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase (MAPK) activation. RESULTS: Lentiviral expression of small hairpin RNAs resulted in robust and specific G(alpha)(12) and G(alpha)(13) knockdown as well as markedly inhibited proliferation, colony formation, and bradykinin-promoted stimulation of cell growth. Analyzing the activation status of all three major MAPK families revealed nonredundant functions of G(alpha)(12) and G(alpha)(13) in SCLC and a marked p42/p44 activation upon G(alpha)(12)/G(alpha)(13) knockdown. In a s.c. tumor xenograft mouse model, G(alpha)(12) or G(alpha)(13) downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, G(alpha)(12)/G(alpha)(13) double knockdown completely abolished H69 tumorigenicity in mice. CONCLUSIONS: G(alpha)(12) and G(alpha)13) exert a complex pattern of nonredundant effects in SCLC, and in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G(12/13) signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment.