Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells.Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted.Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model.Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

The cellular antiviral state mediated by type I interferon (IFN) is the most important host defense mechanism occurring at the early stage of virus infection (15,42,45). IFN binds to the IFN-␣ receptor (IFNAR), which consists of two subunits, IFNAR1 and IFNAR2 (34). The binding of IFN leads to the heterodimerization of the two subunits and the subsequent phosphorylation of two tyrosine kinases, Janus kinase 1 (Jak1) and tyrosine kinase 2 (Tyk2), which are associated with the intracellular domains of the IFNAR (42, 43). Phosphorylated Jak1 and Tyk2, in turn, phosphorylate signal transducer and activator of transcription 1 (STAT1) and STAT2, which are downstream transcriptional factors located in the cytoplasm (9). Once phosphorylated, STAT1 and STAT2 form a trimeric complex with the DNA binding protein, IFN regulatory factor 9, termed IFN-stimulated gene factor 3 (ISGF3) (20,27). ISGF3 then translocates to the nucleus, where it binds to specific promoter elements of IFN-inducible genes (the IFNstimulated response element) and induces the expression of hundreds of IFN-inducible genes that have antiviral and immunoregulatory functions (10,14). However, IFN does not always induce the antiviral response effectively. The efficacy of IFN can be limited by anti-IFN proteins encoded in viral genomes or by host cellular suppressors regulating IFN signaling (24,28,50). Even IFN-sensitive viruses (not armed with anti-

During neurogenesis, proliferating neural precursor cells (NPC) exit the cell cycle and differentiate into postmitotic neurons. The proteins that regulate cell survival through the stages of differentiation, however, are still poorly understood. Here, we examined the roles of the anti-apoptotic Bcl-2 proteins, Mcl-1 and Bcl-xL, in promoting survival as cells progress through the stages of neurogenesis in the mouse embryonic central nervous system. We used Nestin-mediated, nervous systemspecific conditional deletion of mcl-1, bcl-x or both to identify their distinct and overlapping roles. Individual conditional deletion of mcl-1 (MKO) and bcl-x (BKO) suggested sequential roles in promoting cell survival during developmental neurogenesis. In the MKO embryo, apoptosis begins at embryonic day 10 (E10) in the proliferating NPC population throughout the entire developing nervous system. In the BKO embryo, apoptosis begins later at E11 within the postmitotic neuron populations. In the double (mcl-1 and bcl-x) conditional knockout (DKO), cell death extended throughout both proliferating and non-proliferating cell populations resulting in embryonic lethality at E12, earlier than in either the MKO or BKO. Apoptotic cell death of the entire central nervous system in the DKO demonstrates that both genes are necessary for cell survival during developmental neurogenesis. To determine whether Mcl-1 and Bcl-xL have overlapping anti-apoptotic roles during neurogenesis, we examined the impact of gene dosage. Loss of a single bcl-x allele in the MKO embryo exasperated apoptotic cell death within the NPC population revealing a novel anti-apoptotic role for Bcl-xL in proliferating NPCs. Cells were rescued from apoptosis in both the MKO and BKO embryos by breeding with the Bax null mouse line indicating that Mcl-1 and Bcl-xL have a common pro-apoptotic target during developmental neurogenesis. Taken together, these findings demonstrate that Mcl-1 and Bcl-xL are the two essential anti-apoptotic Bcl-2 proteins required for the survival of the developing mammalian nervous system.

Manipulation of body weight set point may be an effective weight loss and maintenance strategy as the homeostatic mechanism governing energy balance remains intact even in obese conditions and counters the effort to lose weight. However, how the set point is determined is not well understood. We show that a single injection of rapamycin (RAP), an mTOR inhibitor, is sufficient to shift the set point in rats. Intraperitoneal RAP decreased food intake and daily weight gain for several days, but surprisingly, there was also a long-term reduction in body weight which lasted at least 10 weeks without additional RAP injection. These effects were not due to malaise or glucose intolerance. Two RAP administrations with a two-week interval had additive effects on body weight without desensitization and significantly reduced the white adipose tissue weight. When challenged with food deprivation, vehicle and RAP-treated rats responded with rebound hyperphagia, suggesting that RAP was not inhibiting compensatory responses to weight loss. Instead, RAP animals defended a lower body weight achieved after RAP treatment. Decreased food intake and body weight were also seen with intracerebroventricular injection of RAP, indicating that the RAP effect is at least partially mediated by the brain. In summary, we found a novel effect of RAP that maintains lower body weight by shifting the set point long-term. Thus, RAP and related compounds may be unique tools to investigate the mechanisms by which the defended level of body weight is determined; such compounds may also be used to complement weight loss strategy.

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1(-/-) MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

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Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

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