Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

No abstract

Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2␣ kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2␣ kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2␣ kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2␣ kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2␣ kinases and p53 with implications in cell proliferation and tumorigenesis.The tumor suppressor p53 is a transcription factor mutated in ϳ50% of human cancers (1). In normal cells, p53 plays a pivotal role in controlling cell cycle, apoptosis, and DNA repair in response to various forms of genotoxic stress (2, 3). The regulation of p53 is complex and occurs mainly at the post-translational level (4). This is mediated by various post-translational modifications, such as phosphorylation and acetylation, which contribute to its stabilization and activation (5). The stability of p53 is regulated by its interaction with Hdm2 (human Mdm2), an E3-ubiquitin ligase that acts as an antagonist limiting p53 tumor suppressor function (6). Both p53 and Hdm2 are in an autoregulatory feedback loop in which p53 induces Hdm2 expression at the transcriptional level. The Hdm2 protein then binds to and ubiquitinates p53 in the nucleus, a process that allows the nuclear export and the cytoplasmic proteasome-dependent degradation of the tumor suppressor (6). In addition to Hdm2, other ubiquitin ligases, such as COP1 (7) and Pirh2 (8), have been shown to disrupt p53 stability. However, compared with Hdm2, little is currently known about how these ligases act on p53 (9).The majority of stress responses that activate p53 require its nuclear accumulation and function (10). This is mediated mainly through inactivation of the Hdm2-dependent degradation pathway as well as through interactions with nuclear proteins that promote post-translational modifications of p53 leading to its stabilization and activation (10). The current interest in p53 is underscored by the tremendous therapeutic benefits of its reactivation in cancer cells. Small molecules or peptides that restore the function of mutant p53 proteins have a great anti-tumor potential by enhancing the apoptotic sensitivity of tumor cells (11-13). Because p53 activity is influenced by many factors, targeting of proteins that regulate p53 function may...

Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs.

Glucose deficiency leads to the induction of eIF2α phosphorylation at serine 51, which results in a global inhibition of protein synthesis. Phosphorylation of eIF2α is an adaptive process that establishes a cytoprotective state in glucose-deficient cells, with possible implications in biological responses that interfere with glucose metabolism.

Cyclin D1 plays a critical role in controlling the G 1 /S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2␣ (eIF2␣) kinases. We examined the effect of activation of the eIF2␣ kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2␣ phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3␤ and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2␣ phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2␣ phosphorylation during short, but not prolonged, periods of stress.

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

Widespread remodeling of the transcriptome is a signature of cancer; however, little is known about the post-transcriptional regulatory factors, including RNA-binding proteins (RBPs) that regulate mRNA stability, and the extent to which RBPs contribute to cancer-associated pathways. Here, by modeling the global change in gene expression based on the effect of sequence-specific RBPs on mRNA stability, we show that RBP-mediated stability programs are recurrently deregulated in cancerous tissues. Particularly, we uncovered several RBPs that contribute to the abnormal transcriptome of renal cell carcinoma (RCC), including PCBP2, ESRP2, and MBNL2. Modulation of these proteins in cancer cell lines alters the expression of pathways that are central to the disease and highlights RBPs as driving master regulators of RCC transcriptome. This study presents a framework for the screening of RBP activities based on computational modeling of mRNA stability programs in cancer and highlights the role of post-transcriptional gene dysregulation in RCC.