Cytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. Here we report that CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, we identify CUTL1 as a transcriptional target of transforming growth factor beta and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression.

Patients with small cell lung cancer (SCLC) die because of chemoresistance. Fibroblast growth factor-2 (FGF-2) increases the expression of antiapoptotic proteins, XIAP and Bcl-X L , and triggers chemoresistance in SCLC cells. Here we show that these effects are mediated through the formation of a specific multiprotein complex comprising B-Raf, PKCe and S6K2. S6K1, Raf-1 and other PKC isoforms do not form similar complexes. RNAi-mediated downregulation of B-Raf, PKCe or S6K2 abolishes FGF-2-mediated survival. In contrast, overexpression of PKCe increases XIAP and Bcl-X L levels and chemoresistance in SCLC cells. In a tetracycline-inducible system, increased S6K2 kinase activity triggers upregulation of XIAP, Bcl-X L and prosurvival effects. However, increased S6K1 kinase activity has no such effect. Thus, S6K2 but not S6K1 mediates prosurvival/chemoresistance signalling.

SummaryCancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

The p90 ribosomal S6 kinases (RSK) are implicated in various cellular processes, including cell proliferation, survival, migration, and invasion. In cancer, RSKs modulate cell transformation, tumorigenesis, and metastasis. Indeed, changes in the expression of RSK isoforms have been reported in several malignancies, including breast, prostate, and lung cancers. Four RSK isoforms have been identified in humans on the basis of their high degree of sequence homology. Although this similarity suggests some functional redundancy between these proteins, an increasing body of evidence supports the existence of isoform-based specificity among RSKs in mediating particular cellular processes. This review briefly presents the similarities between RSK family members before focusing on the specific function of each of the isoforms and their involvement in cancer progression. Cancer Res; 73(17); 5301-8. Ó2013 AACR.

A poptosis, or programmed cell death, is important for the normal development of organisms and maintaining tissue homeostasis. Misregulation of apoptosis may lead to neurodegenerative disorders when apoptosis is increased and tumor formation when apoptosis is inhibited; thus, the pathway is tightly controlled, with both pro-and antiapoptotic factors playing a role. Of particular interest are members of the inhibitor of apoptosis (IAP) and Bcl-2 families of proteins that intercept virtually all apoptotic signals in the cell.X chromosome-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family; it directly interacts with and inhibits caspases 3, 7, and 9 and is therefore a key regulator of apoptosis (20). In contrast, Bcl-x L controls apoptosis by maintaining mitochondrial membrane homeostasis (14). Interestingly, both the XIAP and Bcl-x L mRNAs contain an internal ribosome entry site (IRES) that allows them to be translated during cellular stress by a cap-independent mechanism when cap-dependent translation is inhibited, which is necessary for their protective roles in the cell (4,11,16,24,49). Under normal growth conditions, translation of cellular mRNAs occurs through a cap-dependent mechanism that requires interaction of specific initiation factors (such as eukaryotic initiation factor 4E [eIF4E]) with the 5= cap of the mRNA, followed by recruitment of ribosomal subunits, recognition of the AUG start codon, and commencement of polypeptide chain elongation (reviewed in reference 26). However, certain cellular stresses such as nutrient deprivation, hypoxia, or low-dose irradiation cause attenuation of cap-dependent translation, and yet, under these conditions, a sizeable proportion of cellular mRNAs, perhaps as much as 10%, have been shown to be translated by a cap-independent mechanism, such as through an IRES (23,28,39). IRES elements are located within the 5= untranslated region (UTR) of some cellular mRNAs and are believed to recruit the ribosome directly, thereby bypassing the requirement for the mRNA 5= cap and eIF4E. Moreover, while IRES-dependent translation requires some canonical translation initiation factors, most (if not all) cellular IRES elements require the activity of auxiliary RNA binding proteins that function as IRES trans-acting factors (ITAFs) (reviewed in references 28 and 32). The mechanism by which ITAFs function is poorly understood, and since different IRESs require different sets of ITAFs, elucidating the identity of all known ITAFs has proven challenging. Furthermore, it has been shown that ITAFs can function as either positive or negative regulators of IRES-mediated translation. For example, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was shown to act as a repressor of XIAP IRES-mediated translation (33), whereas La autoantigen (19), hnRNPC1 and hnRNPC2 (18), and HuR (11) have all been shown to have a stimulating effect on XIAP IRES-mediated translation.We have previously demonstrated that treatment of small-cell lung cancer cells with fibroblast gr...

Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2-induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham-treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3′-deoxy-3′-