Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9.

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.

In the absence of its cytosolic domain, the CD28 molecule still contributes to T cell activation.

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.

Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation.

Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1-/- MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA.

The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells.

One of the most exciting areas of current research in the cannabinoid field is the study of the potential application of these compounds as antitumoral drugs. Here, we describe the signaling pathway that mediates cannabinoid-induced apoptosis of tumor cells. By using a wide array of experimental approaches, we identify the stress-regulated protein p8 (also designated as candidate of metastasis 1) as an essential mediator of cannabinoid antitumoral action and show that p8 upregulation is dependent on de novo-synthesized ceramide. We also observe that p8 mediates its apoptotic effect via upregulation of the endoplasmic reticulum stress-related genes ATF-4, CHOP, and TRB3. Activation of this pathway may constitute a potential therapeutic strategy for inhibiting tumor growth.

A review of kinases implicated in pancreatic cancer.

The current 5-year survival rate of pancreatic cancer is about 3% and the median survival less than 6 months because the chemotherapy and radiation therapy presently available provide only marginal benefit. Clearly, pancreatic cancer requires new therapeutic concepts. Recently, the kinase inhibitors imatinib and gefitinib, developed to treat chronic myelogenous leukaemia and breast cancer, respectively, gave very good results. Kinases are deregulated in many diseases, including cancer. Given that phosphorylation controls cell survival signalling, strategies targeting kinases should obviously improve cancer treatment. The purpose of this review is to summarize the present knowledge on kinases potentially usable as therapeutic targets in the treatment of pancreatic cancer. All clinical trials using available kinase inhibitors in monotherapy or in combination with chemotherapeutic drugs failed to improve survival of patients with pancreatic cancer. To detect kinases relevant to this disease, we undertook a systematic screening of the human kinome to define a 'survival kinase' catalogue for pancreatic cells. We selected 56 kinases that are potential therapeutic targets in pancreatic cancer. Preclinical studies using combined inhibition of PAK7, MAP3K7 and CK2 survival kinases in vitro and in vivo showed a cumulative effect on apoptosis induction. We also observed that these three kinases are rather specific of pancreatic cancer cells. In conclusion, if kinase inhibitors presently available are unfortunately not efficient for treating pancreatic cancer, recent data suggest that inhibitors of other kinases, involved more specifically in pancreatic cancer development, might, in the future, become interesting therapeutic targets.

Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance.

Except for gemcitabine, chemotherapeutic agents are ineffective with pancreatic adenocarcinoma because this cancer is resistant to apoptosis induction. Involvement of specific kinases in such resistance is likely. We developed a systematic strategy to screen the human kinome and select kinases whose inhibition in pancreatic cancer cells can increase 1) spontaneous apoptosis or 2) gemcitabine-induced apoptosis. The pancreatic adenocarcinoma cell line MiaPaCa-2 was transfected with 645 pairs of siRNAs directed to all human kinases. The same experiment was conducted in cells treated with 150 microM gemcitabine. Apoptosis was measured after 2 days and the results were normalized for cell viability. A panel of 56 kinases whose inhibition increased spontaneous apoptosis by at least 50% was established. Ten of them gave similar results on Panc1 and BxPC3 pancreatic adenocarcinoma cell lines. A panel of 83 kinases whose inhibition increased gemcitabine-induced apoptosis by 50% or more was also established. Twelve kinases appeared in both panels. A cumulative increase in apoptosis was observed when inhibiting simultaneously several kinases. Such a systematic approach allowed characterization of all kinases involved in pancreatic cancer cell survival and resistance to gemcitabine. Inhibitors of these kinases, used alone or in combination, might improve the treatment of pancreatic adenocarcinoma.

p8 is a new target of gemcitabine in pancreatic cancer cells.

Gemcitabine is the only available chemotherapeutic treatment of pancreatic cancers. It is, however, moderately effective, showing a tumor response rate of only 12%. The aim of this work was to identify new pathways involved in the resistance of pancreatic cancer cells to gemcitabine, in the hope of developing new adjuvant strategies to enhance its therapeutic efficacy. Comparison of gene expression patterns of five human pancreatic cancer cell lines showing different degrees of resistance to gemcitabine revealed specific overexpression of several genes in the most resistant. One of them encoded the antiapoptotic p8 protein. We found that (a) knocking down p8 expression in gemcitabine-resistant cells promoted cell death and increased caspase-3 activity; (b) forced overexpression of p8 in gemcitabine-sensitive cells increased their resistance to gemcitabine-induced apoptosis; and (c) gemcitabine down-regulated p8 mRNA expression. These results suggest that, in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8. Hence, targeting the p8-associated pathway could be a new adjuvant therapy improving the response of patients with pancreatic cancer to gemcitabine treatment.

Regulation of apoptosis by the p8/prothymosin alpha complex.

p8 is a small-stress protein involved in several cellular functions including apoptosis. To identify its putative partners, we screened a HeLa cDNA library by using the two-hybrid technique and found that p8 binds the antiapoptotic protein prothymosin alpha (ProTalpha). Fluorescence spectroscopy, circular dichroism, and NMR spectroscopy showed that p8 and ProTalpha formed a complex. Binding resulted in important changes in the secondary and tertiary structures of the proteins. Because p8 and ProTalpha form a complex, they could act in concert to regulate the apoptotic cascade. We induced apoptosis in HeLa cells by staurosporine treatment and monitored the effects of knocking down p8 and/or ProTalpha or overexpressing p8 and/or ProTalpha on caspase 3/7 and 9 activities and on cell death. Transfecting ProTalpha or p8 small interfering RNAs increased the activities of both caspases and the number of apoptotic nuclei. However, transfecting both small interfering RNAs resulted in no further increase. Overexpressing p8 or ProTalpha did not alter caspase activities, whereas overexpressing both resulted in a significant reduction of caspase activities. These results strongly suggest that the antiapoptotic response of HeLa cells upon staurosporine treatment requires expression of both p8 and ProTalpha.