Cancer-associated cachexia (CAC) is a wasting syndrome characterized by systemic inflammation, body weight loss, atrophy of white adipose tissue (WAT) and skeletal muscle. Limited therapeutic options are available and the underlying mechanisms are poorly defined. Here we show that a phenotypic switch from WAT to brown fat, a phenomenon termed WAT browning, takes place in the initial stages of CAC, before skeletal muscle atrophy. WAT browning is associated with increased expression of uncoupling protein 1 (UCP1), which uncouples mitochondrial respiration toward thermogenesis instead of ATP synthesis, leading to increased lipid mobilization and energy expenditure in cachectic mice. Chronic inflammation and the cytokine interleukin-6 increase UCP1 expression in WAT, and treatments that reduce inflammation or β-adrenergic blockade reduce WAT browning and ameliorate the severity of cachexia. Importantly, UCP1 staining is observed in WAT from CAC patients. Thus, inhibition of WAT browning represents a promising approach to ameliorate cachexia in cancer patients.

The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling.

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.

USA 3Corresponding author N.M.T.van der Lugt and J.Domen contributed equally to this workThe Pim-1 proto-oncogene is one of the most potent collaborators of the myc proto-oncogenes in inducing lymphomagenesis in mice. Contrary to the profound effects when overexpressed in vivo, Pim-l-deficient mice showed only subtle phenotypic alterations, which could indicate the presence of redundantly acting genes. In line with this, a PCR-based screen has led to the identification of a closely homologous gene, Pim-2. The X-linked Pim-2 gene is 53% identical to Pim-1 at the amino acid level and shares substrate preference and the usage of non-AUG initiation codons with Pim-1. We have used these data to test whether the strong synergistic interaction between Pim-1 and c-myc can be utilized to gain access to Pim-1 compensatory pathways. We reasoned that, upon proviral tagging in compound mutant mice (Eji-myc/Pim-L'-1 mice), the selective advantage of cells carrying provirally activated genes, that act downstream from or parallel to Pim-1, would increase. We show here that this is the case. A dramatic increase (from 15 to 80%) was found in the frequency of proviral activation of the Pim-2 gene. These data show that the described strategy of 'complementation tagging' represents a powerful new tool to identify components of pathways involved in processes as complex as multistep tumorigenesis.

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1-deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Upregulation of Mcl-1 was also due to increased transcription that involved the IRE1A and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress. [Cancer Res 2008;68(16):6708-17]

Purpose: Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a smallmolecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL, and BCL-w and shows promise for treating leukemia, lymphoma, and small-cell lung cancer. Melanoma cells are insensitive to ABT-737, but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound.Experimental Design: The efficacy of MCL-1 and BFL-1 inhibition for sensitizing melanoma cells to ABT-737 was investigated by short hairpin RNA-mediated knockdown or overexpression of their antagonist NOXA in two-dimensional cell culture, a three-dimensional organotypic spheroid model, and an in vivo model.Results: MCL-1 downregulation or NOXA overexpression strongly sensitized melanoma cells to ABT-737 in vitro. NOXA-inducing cytotoxic drugs also strongly sensitized melanomas to ABT-737 but, surprisingly, not vice versa. The drugs most suitable are not necessarily those normally used to treat melanoma. Resistance to ABT-737 occurred quickly in three-dimensional melanoma spheroids through reduced NOXA expression, although experiments with both xenografts and three-dimensional spheroids suggest that penetration of ABT-737 into tumor masses may be the principal limitation, which may be obviated through use of more diffusible BH3-mimetics.Conclusion: Sensitization of tumors to BH3-mimetics by cytotoxic drugs that induce NOXA is a therapeutic strategy worth exploring for the treatment of melanoma and other solid cancers. Clin Cancer Res; 18(3); 783-95. Ó2011 AACR.

Members of the multidrug resistance -associated protein (MRP) family of transporters are believed to contribute to cytotoxic drug resistance and chemotherapy failure. We observed frequent MRP4 overexpression in aggressive primary neuroblastoma, a disease for which we have previously shown MRP1 to be a prognostic indicator. High MRP4 expression correlated with MYCN oncogene amplification and was significantly associated with poor clinical outcome. Although MRP4 is known to transport some nucleoside analogues, it has not previously been associated with resistance to drugs used to treat solid tumors. We now show that it mediates substantial resistance in vitro to the topoisomerase I poison irinotecan/CPT-11 and its active metabolite SN-38. These results suggest that MRP4 will be a useful prognostic marker for neuroblastoma and that clinical trials of irinotecan as a neuroblastoma treatment should monitor MRP4 expression. The same may be true for other tumor types expressing high levels of the transporter. [Mol Cancer Ther 2005;4(4):547 -53]

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