Lakshmi Raj

Selective killing of cancer cells by a small molecule targeting the stress response to ROS

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

Hzf Determines Cell Survival upon Genotoxic Stress by Modulating p53 Transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.

Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress

The Ser/Thr Rho kinase 1 (ROCK1) is known to play major roles in a wide range of cellular activities, including those involved in tumor metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the PI(3)kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex, without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy.

Identification of ROCK1 as an upstream activator of the JIP-3 to JNK signaling axis in response to UVB damage

The Rho-associated kinase ROCK1 mediates the cellular response to UV radiation. A ROCK in Stressful Times Exposure of cells to ultraviolet B (UVB) radiation results in damage to DNA and the generation of mutations that can give rise to unchecked cellular proliferation. Thus, it is critically important to the prevention of skin cancers that the response to UVB-induced damage is to trigger apoptosis in the affected cells. In UVB-exposed cells, activation of members of the mitogen-activated protein kinase (MAPK) family, such as c-Jun N-terminal kinase (JNK), mediates this apoptotic response, but how UVB radiation activates signaling pathways upstream of JNK is unclear. Ongusaha et al. now show that exposure of keratinocytes to UVB radiation activates Rho-associated kinase 1 (ROCK1), which phosphorylates and activates the scaffold protein JNK-interacting protein 3 (JIP-3), which in turn recruits and activates JNK. Mice deficient in ROCK1 failed to mount as efficient an apoptotic response to UVB irradiation as did their wild-type counterparts, suggesting that ROCK1 is a critical early player in the response to UVB irradiation. Although apoptosis triggered by ultraviolet B (UVB)–mediated activation of the c-Jun N-terminal kinase (JNK) pathway is mediated by both intrinsic and extrinsic pathways, the mechanism of initiation of JNK activation remains obscure. Here, we report the characterization of the JNK-interacting protein 3 (JIP-3) scaffolding protein as an interacting partner of Rho-associated kinase 1 (ROCK1), as determined by tandem affinity protein purification. Upon UVB-induced stress in keratinocytes, ROCK1 was activated, bound to JIP-3, and activated the JNK pathway. Moreover, phosphorylation of JIP-3 by ROCK1 was crucial for the recruitment of JNK. Inhibition of the activity of ROCK1 in keratinocytes resulted in decreased activation of the JNK pathway and thus a reduction in apoptosis. ROCK1+/− mice exhibited decreased UVB-mediated activation of JNK and apoptosis relative to wild-type mice. Our findings present a new molecular mechanism by which ROCK1 functions as a UVB sensor that regulates apoptosis, an important event in the prevention of skin cancer.

Msx1 and Tbx2 antagonistically regulate Bmp4 expression during the bud-to-cap stage transition in tooth development

Bmp4 expression is tightly regulated during embryonic tooth development, with early expression in the dental epithelial placode leading to later expression in the dental mesenchyme. Msx1 is among several transcription factors that are induced by epithelial Bmp4 and that, in turn, are necessary for the induction and maintenance of dental mesenchymal Bmp4 expression. Thus, Msx1-/- teeth arrest at early bud stage and show loss of Bmp4 expression in the mesenchyme. Ectopic expression of Bmp4 rescues this bud stage arrest. We have identified Tbx2 expression in the dental mesenchyme at bud stage and show that this can be induced by epithelial Bmp4. We also show that endogenous Tbx2 and Msx1 can physically interact in mouse C3H10T1/2 cells. In order to ascertain a functional relationship between Msx1 and Tbx2 in tooth development, we crossed Tbx2 and Msx1 mutant mice. Our data show that the bud stage tooth arrest in Msx1-/- mice is partially rescued in Msx1-/-;Tbx2+/- compound mutants. This rescue is accompanied by formation of the enamel knot (EK) and by restoration of mesenchymal Bmp4 expression. Finally, knockdown of Tbx2 in C3H10T1/2 cells results in an increase in Bmp4 expression. Together, these data identify a novel role for Tbx2 in tooth development and suggest that, following their induction by epithelial Bmp4, Msx1 and Tbx2 in turn antagonistically regulate odontogenic activity that leads to EK formation and to mesenchymal Bmp4 expression at the key bud-to-cap stage transition.

Corrigendum: Selective killing of cancer cells by a small molecule targeting the stress response to ROS.

This corrects the article DOI: 10.1038/nature10167

A Network of Transcription Factors Operates during Early Tooth Morphogenesis

ABSTRACT Improving the knowledge of disease-causing genes is a unique challenge in human health. Although it is known that genes causing similar diseases tend to lie close to one another in a network of protein-protein or functional interactions, the identification of these protein-protein networks is difficult to unravel. Here, we show that Msx1, Snail, Lhx6, Lhx8, Sp3, and Lef1 interact in vitro and in vivo, revealing the existence of a novel context-specific protein network. These proteins are all expressed in the neural crest-derived dental mesenchyme and cause tooth agenesis disorder when mutated in mouse and/or human. We also identified an in vivo direct target for Msx1 function, the cyclin D-dependent kinase (CDK) inhibitor p19ink4d, whose transcription is differentially modulated by the protein network. Considering the important role of p19ink4d as a cell cycle regulator, these results provide evidence for the first time of the unique plasticity of the Msx1-dependent network of proteins in conferring differential transcriptional output and in controlling the cell cycle through the regulation of a cyclin D-dependent kinase inhibitor. Collectively, these data reveal a novel protein network operating in the neural crest-derived dental mesenchyme that is relevant for many other areas of developmental and evolutionary biology.

Retraction Note: Selective killing of cancer cells by a small molecule targeting the stress response to ROS

This Letter is being retracted owing to issues with Fig. 1d and Supplementary Fig. 31b, and the unavailability of original data for these figures that raise concerns regarding the integrity of the figures. Nature published two previous corrections related to this Letter1,2. These issues in aggregate undermine the confidence in the integrity of this study. Authors Michael Foley, Monica Schenone, Nicola J. Tolliday, Todd R. Golub, Steven A. Carr, Alykhan F. Shamji, Andrew M. Stern and Stuart L. Schreiber agree with the Retraction. Authors Lakshmi Raj, Takao Ide, Aditi U. Gurkar, Anna Mandinova and Sam W. Lee disagree with the Retraction. Author Xiaoyu Li did not respond.