Olivier E. Pardo

Yes-associated protein (YAP) functions as a tumor suppressor in breast

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.

FGF‐2 protects small cell lung cancer cells from apoptosis through a complex involving PKCε, B‐Raf and S6K2

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‐XL, 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, PKCε and S6K2. S6K1, Raf‐1 and other PKC isoforms do not form similar complexes. RNAi‐mediated downregulation of B‐Raf, PKCε or S6K2 abolishes FGF‐2‐mediated survival. In contrast, overexpression of PKCε increases XIAP and Bcl‐XL levels and chemoresistance in SCLC cells. In a tetracycline‐inducible system, increased S6K2 kinase activity triggers upregulation of XIAP, Bcl‐XL and prosurvival effects. However, increased S6K1 kinase activity has no such effect. Thus, S6K2 but not S6K1 mediates prosurvival/chemoresistance signalling.

Fibroblast Growth Factor 2-Mediated Translational Control of IAPs Blocks Mitochondrial Release of Smac/DIABLO and Apoptosis in Small Cell Lung Cancer Cells

ABSTRACT The mitochondrial release of cytochrome c and Smac/DIABLO has been implicated in the activation of apoptosis in response to cell stress. Smac promotes cytochrome c-induced activation of caspases by sequestering the inhibitor of apoptosis protein (IAP) family of potent caspase suppressors. Differential release from mitochondria of cytochrome c and Smac can occur, but the underlying mechanism and physiological significance of this are unclear. Here we show that the mechanism by which fibroblast growth factor 2 (FGF-2) protects small cell lung cancer (SCLC) cells from etoposide-induced cell death involves inhibition of Smac release but not of cytochrome c release. This process is MEK dependent and correlates with an increased expression of XIAP and cellular IAP-1, mediated principally through translational regulation. Exogenous expression of XIAP is sufficient to inhibit caspase 9 activation, Smac release, and cell death induced by etoposide. Prevention of the FGF-2-promoted increase in levels of functional IAPs by RNA interference or the cell-permeant Smac amino-terminal peptide blocked FGF-2-induced protection. FGF-2 can thus protect SCLC cells from chemotherapeutic drugs by modulating IAP levels via posttranscriptional regulation, providing a mechanism for postmitochondrial survival signaling by the MEK/mitogen-activated protein kinase pathway.

AKT/mTOR Pathway Activation and BCL-2 Family Proteins Modulate the Sensitivity of Human Small Cell Lung Cancer Cells to RAD001

Purpose: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. Experimental Design: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. Results: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor–stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2–specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. Conclusion: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

An siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis

We performed a kinome-wide siRNA screen and identified 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) silencing increased, whereas RSK2 and RSK4 downregulation inhibited cell motility. In a secondary collagen-based three-dimensional invasion screen, 38 of our hits cross-validated, including RSK1 and RSK4. In two further lung cancer cell lines, RSK1 but not RSK4 silencing showed identical modulation of cell motility. We therefore selected RSK1 for further investigation. Bioinformatic analysis followed by co-immunoprecipitation-based validation revealed that the actin regulators VASP and Mena interact with RSK1. Moreover, RSK1 phosphorylated VASP on T278, a site regulating its binding to actin. In addition, silencing of RSK1 enhanced the metastatic potential of these cells in vivo using a zebrafish model. Finally, we investigated the relevance of this finding in human lung cancer samples. In isogenically matched tissue, RSK1 was reduced in metastatic versus primary lung cancer lesions. Moreover, patients with RSK1-negative lung tumours showed increased number of metastases. Our results suggest that the findings of our high-throughput in vitro screen can reliably identify relevant clinical targets and as a proof of principle, RSK1 may provide a biomarker for metastasis in lung cancer patients.

Novel cross talk between MEK and S6K2 in FGF-2 induced proliferation of SCLC cells

Here, we show that fibroblast growth factor-2 (FGF-2) induces proliferation of H-510 and H-69 small cell lung cancer (SCLC) cells. However, the optimal response to FGF-2 was obtained at 10-fold lower concentrations in H-510 cells. This correlated with the selective activation of the mitogen-activated protein kinase kinase (MEK) pathway in H-510, but not H-69 cells. Moreover, inhibition of MEK with PD098059 blocked FGF-2-induced proliferation in H-510 cells only. Similarly, ribosomal protein S6 kinase 2 (S6K2), a recently identified homologue of S6K1 was activated by FGF-2 in H-510, but not H-69 cells. This activation was independent of phosphatidylinositol-3 kinase, but was sensitive to inhibition of the MEK pathway. These data suggest that S6K2 is a novel downstream target of MEK. The potency of FGF-2 in H-510 cells might reflect this additional MEK/S6K2 signalling. In contrast to S6K2, S6K1 was activated in both SCLC cell lines. Inhibition of the mammalian target of rapamycin with 10 ng/ml rapamycin blocked S6K1 activation and proliferation of both lines. However, even at 100 ng/ml, rapamycin only partially inhibited S6K2. Strikingly, this correlated with inhibition of MEK signalling. Our data indicate that S6K1, and possibly S6K2, are involved in FGF-2-induced SCLC cell growth, a notion supported by the overexpression and higher baseline activity of both isoforms in SCLC lines, as compared to normal human type-II pneumocytes.

Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

ABSTRACT Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.

The p90 RSK Family Members: Common Functions and Isoform Specificity

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.

Involvement of Heterogeneous Ribonucleoprotein F in the Regulation of Cell Proliferation via the Mammalian Target of Rapamycin/S6 Kinase 2 Pathway

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.

S6K2: The Neglected S6 Kinase Family Member

S6 kinase 2 (S6K2) is a member of the AGC kinases super-family. Its closest homolog, S6K1, has been extensively studied along the years. However, due to the belief in the community that the high degree of identity between these two isoforms would translate in essentially identical biological functions, S6K2 has been largely neglected. Nevertheless, recent research has clearly highlighted that these two proteins significantly differ in their roles in vitro as well as in vivo. These findings are significant to our understanding of S6 kinase signaling and the development of therapeutic strategies for several diseases including cancer. Here, we will focus on S6K2 and review the protein–protein interactions and specific substrates that determine the selective functions of this kinase.