In an attempt to identify molecules that clearly reflect the oncogenic role of cell signaling pathways in human tumors, we propose a concept we term ''funnel factor'', a factor where several oncogenic signals converge and drive the proliferative signal downstream. In studies done in various tumor types, the expression of key cell signaling factors, including Her1 and Her2 growth factor receptors, as well as the RAS-RAF-mitogenactivated protein kinase and the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathways was correlated with the associated clinicopathologic characteristics of these tumors. The downstream factors p70, S6, 4E-binding protein 1 (4E-BP1), and eukaryotic translation initiation factor 4E, which play a critical role in the control of protein synthesis, survival, and cell growth, were also analyzed. We found that phosphorylated 4E-BP1 (p-4E-BP1) expression in breast, ovary, and prostate tumors is associated with malignant progression and an adverse prognosis regardless of the upstream oncogenic alterations. Thus, p-4E-BP1 seems to act as a funnel factor for an essential oncogenic capability of tumor cells, self-sufficiency in growth signals, and could be a highly relevant molecular marker of malignant potential. Further investigation into this concept may identify additional funnel factors in the oncogenic pathways and provide potential therapeutic targets. [Cancer Res 2007;67(16):7551-5]

Purpose: The control of senescence and its biochemical pathways is a crucial factor for understanding cell transformation. In a large RNA interference screen, the RSK4 gene was found to be related to p53-dependent arrest. The purpose of the present study was to investigate the potential role of RSK4 as a tumor suppressor gene. Experimental Design: RSK4 expression was determined by quantitative real-time PCR and immunoblot in 30 colon and 20 renal carcinomas, and in 7 colon adenomas. Two HCT116 colon carcinoma cell lines (p53 wt and p53 null), IMR90 human fibroblasts, and E1A-expressing IMR90 cells were infected with RSK4 cDNA and/or shRNA. RSK4 expression levels were analyzed in HCT116 p53 wt or p53 null and IMR90 after senescence induction by quantitative real-time PCR and Western blot.Results: The RSK4 gene was down-regulated in 27 of 30 colon carcinomas (P < 0.001), 16 of 20 renal cell carcinomas (P < 0.01), and 6 of 7 colon adenomas (P < 0.01). In vitro overexpression of RSK4 induced cell arrest and senescence features in normal fibroblasts and malignant colon carcinoma cell lines. Interestingly, in these cell lines RSK4 mRNA levels were increased both in replicative and stress-induced senescence. Moreover, IMR90 partially immortalized by RSK4 shRNA and HCT116 with this short hairpin RNA were more resistant to cisplatin treatment. Finally, cells expressing E1A or Rb short interfering RNA were resistant to RSK4-mediated senescence. Conclusion: These results support the concept that RSK4 may be an important tumor suppressor gene by modulating senescence induction and contributing to cell proliferation control in colon carcinogenesis and renal cell carcinomas.Cellular senescence can be defined as an irreversible arrest of cell proliferation. It is activated in response to various types of stress, including oxidative stress (e.g. hydrogen peroxide treatment), oncogene activities, DNA damage, and treatment with DNA-damaging agents (e.g. doxorubicin or cisplatin), among others. This is called accelerated or stress-induced senescence. Moreover, senescence can occur as a result of telomere shortening after multiple cell divisions, a process known as replicative senescence (1, 2). Once cells enter senescence, they stop growing and develop dramatic morphologic changes, such as a flat, enlarged morphology, as well as metabolic changes (3 -7).Two main pathways are reported to be involved in senescence, p16INK4a /Rb and p19 ARF /p53, which are considered to be the main activators of senescence (7,8). P16 activates Rb by inhibiting CycD/Cdk4,6. P19 activates p53 by inhibiting MDM2; p53 can be also activated by phosphorylation done by the ATM/ATR and/or Chk1/Chk2 proteins. P53 and Rb can be connected through p21, activated by p53, which, in turn, can activate Rb by inhibiting CycE/Cdk2. Once Rb is activated, it is able to shut down transcription of the E2F target genes, inducing cell growth arrest. In addition, it seems that p53 can activate senescence in human cells independently of Rb (8,9). Whether both these s...

p90 Ribosomal S6 kinase (RSK) 4 is a serine-threonine kinase that belongs to the p90RSK family. RSK4 has been proposed as a tumor suppressor gene, related with anti-invasive activity, inhibition of the RAS-mitogen-activated protein kinase (MAPK) pathway and induction of senescence. Despite the related findings, little is known about RSK4 effectors. In human tumors, RSK4 is downregulated even in some benign lesions, such as colon adenomas and breast papillomas, indicating that RSK4 inhibition could be an early event in cellular transformation. For cells to achieve immortality and transformation, it is believed that they must override senescence. In the present study, we found that when RSK4 is inhibited in vitro using short hairpin RNA technology, cells can bypass stress-induced senescence and oncogene-induced senescence: normal human fibroblasts grew following oxidative stress, induction of DNA damage and KRAS(V12) or BRAF(E600) overexpression. To investigate the RSK4 effectors, we used short hairpin RNA or inhibitor molecules against major senescence mediators. We found that RSK4-induced senescence is mediated through p21, but is independent of p16, p38MAPKs and induction of reactive oxygen species, delimiting RSK4 signaling. These data support the importance of RSK4 for regulating senescence and indicate that downregulation of this kinase could be an important element in facilitating cell transformation.

Abstract.Irvalec ® (elisidepsin trifluoroacetate, PM02734) is a novel marine-derived cyclic peptide belonging to the Kahaladide family of compounds, currently in clinical trials with preliminary evidence of antitumor activity. Previous studies have shown a correlation between elisidepsin sensitivity and expression of the ErbB3 receptor in a panel of NSCLC cell lines. We have studied the effect of elisidepsin on the ErbB3 pathway, characterizing the expression of all members of the ErbB (HER) family of receptors and their main downstream signaling effectors, such as Akt and MAPK. Interestingly, we observed a downregulation of ErbB3 upon elisidepsin treatment that correlates with a reduction in the Akt phosphorylation levels in the most sensitive cell lines, whereas ErbB3 levels are not affected in the less sensitive ones. Also, we observed that the basal levels of ErbB3 protein expression show a significant correlation with cell viability response against elisidepsin treatment in 14 different cell lines. Furthermore, we analyzed the combination of elisidepsin with different chemotherapeutics agents, such as cisplatin, paclitaxel and gemcitabine, in a panel of different breast (MDA-MB-435, MDA-MB-231 and MCF7), lung (HOP62, DV90 and A549) and colorectal cancer cell lines (DLD1 and HT29). IC 50 values for the different drugs were tested. We observed a synergistic effect in all cell lines tested with any chemotherapeutic agent. More importantly, the two in vitro elisidepsin-resistant cell lines (MDA-MB-231 and HOP62) presented a synergistic effect in combination with cisplatin and paclitaxel, respectively. These results provide a rationale for further development of these combinations in an ongoing clinical trial.

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Abstract. Translational control is a crucial component of cancer development and progression. Eukaryotic initiation factor (eIF) 4E mediates eIF4F association with the mRNA 5' cap structure to stimulate cap-dependent translation initiation. The eIF4E-binding protein, 4E-BP1, regulates cap-dependent translation through its phosphorylation at multiple sites. It has been described that some human carcinomas present a high level of p-4E-BP1, not always associated with high levels of p-mTOR. These previous observations suggest that other kinases could be involved in 4E-BP1 phosporylation. Investigation in new kinases that could be implicated in 4E-BP1 phosphorylation and mechanisms that affect 4E-BP1 stability is important to understand the role of eIF4E in cell transformation. In this study, we examined 48 kinases that could be involved in 4E-BP1 phosphorylation and stability. The screening study was based on analysis of 4E-BP1 status after inhibition of these kinases in a breast carcinoma cell line. Several kinases affecting 4E-BP1 stability (LRRK2, RAF-1, p38γ, GSK3β, AMPKα, PRKACA and PRKACB) and 4E-BP1 phosphorylation (CDK1, PDK1, SRC, PRKCB1, PAK2, p38β, PRKCA and CaMKKB) were identified. These findings provide evidence that 4E-BP1 can be regulated and stabilized by multiple kinases implicated in several cell signaling pathways. We focus on the finding that LRRK2 down-regulation was associated with a clearly decreased 4E-BP1 protein (and not with mRNA down-regulation). Importantly, knockdown of LRRK2 associated with high proliferative rate in normal cells and treatment with rapamycin and/or proteosome inhibition suppressed 4E-BP1 protein degradation. These results offer new insights into the regulation of total and phosphorylated 4E-BP1.

The antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. Methods: Twenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in 10 countries/regions. Depending on each pathogen, antibody levels were measured using specific commercial IgG-specific enzyme immunoassay kits or by cell culture neutralization test and guinea pig skin neutralization test. A principal component analysis was performed. Results: For polio and diphtheria (reference markers of the US authorities), all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia, and the USA were positive for titers against globally distributed pathogens or those under vaccination programs in the developed world (parainfluenza, Epstein-Barr, varicella-zoster, influenza B, parvovirus B19, and measles viruses). IVIG from Taiwan and Hong Kong showed low antibody titers for these pathogens but high titers for Pseudomonas aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, and hepatitis E viruses and Streptococcus pneumoniae). IVIGs from Argentina, Spain, Israel, and Czechia showed intermediate antibody concentrations. Conclusion:The antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs, and the prevalence of pathogens in the different countries and regions.

Translational control is a crucial component of cancer development and progression. Eukaryotic initiation factor 4E (eIF4E) mediates eIF4F association with the mRNA 5’ cap structure to stimulate cap-dependent translation initiation. The eIF4E-binding protein, 4E-BP1, regulates cap-dependent translation through its phosphorylation at multiple sites. The search for new kinases that could be implicated in 4E-BP1 phosphorylation, and determination of the mechanisms that affect 4E-BP1 stability are important to understand the role of eIF4E in cell transformation. In this study, we investigate 48 kinases that may be implicated in 4E-BP1 phosphorylation and stability. The screening study was based on analysis of 4E-BP1 status after inhibition of these kinases in a breast carcinoma cell line (MDA-MB 231 cells). With the use of this method, several kinases that affect 4E-BP1 stability (LRRK2, RAF-1, p38γ, GSK3β, AMPKα, PRKACA and PRKACB) and 4E-BP1 phosphorylation (CDK1, PDK1, SRC, PRKCB1, PAK2, p38β PRKCA and CaMKKB) were identified. These novel findings provide evidence that 4E-BP1 can be regulated and stabilized by multiple kinases implicated in several cell signaling pathways. We also found that LRRK2 downregulation affects 4E-BP1 stability and causes degradation of this factor in various cell types. Interestingly, inhibition of proteasome activity by MG132 restored total 4E-BP1 protein levels in cells where LRRK2 was downregulated. In addition, these stabilization events appear to be functionally important for cell growth control in normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2011-1939