An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells

A functional genomic approach to actionable gene fusions for precision oncology

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize similar to 100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology

The identification of phosphatases important for mitosis and replicative stress response

In every cell division cycle, daughter cells carry the same genomic information as their parental cell. Maintenance of the genomic stability requires several checkpoints that function at different stages of the cell cycle. These checkpoints halt cell cycle progression to allow cells to have the time to correct errors. Cells with defective checkpoints may allow errors to accumulate and eventually promote tumorigenesis. Therefore, identifying components of the checkpoints is important for a better understanding of how genome stability is maintained. Protein phosphorylation is important in many cellular processes, including normal cell cycle progression and checkpoint regulation. While protein kinases involved in cell cycle control and checkpoints have been studied extensively, the protein phosphatases involving in the processes are less understood. To identify novel phosphatases important for mitosis and replicative stress responses, RNA interference screens targeting the all phosphatases in the human genome were performed. A short-hairpin RNA library against the human phosphatome was generated. These screens were designed to identify phosphatases that, when depleted, affected mitotic progression and survival after replicative stress. A large number of phosphatases were identified to be required for proper mitotic progression. Several phosphatases, including the non-receptor type protein tyrosine phosphatase PTPN11 (SHP2), were identified to be important for survival after replicative stress. Cells were more susceptible to hydroxyurea-induced cell death in the absence of SHP2. Furthermore, SHP2 is required for cell survival after other DNA damaging agents, indicating that SHP2 is critical for different DNA stresses. Collectively, these studies underscore the importance of protein phosphatases in cell cycle control

Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCFβ-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis ▿ †

Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G2 arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCFβ-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed

Novel functions of the phosphatase SHP2 in the DNA replication and damage checkpoints.

Replication stress- and DNA damage-induced cell cycle checkpoints are critical for maintaining genome stability. To identify protein phosphatases involved in the activation and maintenance of the checkpoints, we have carried out RNA interference-based screens with a human phosphatome shRNA library. Several phosphatases, including SHP2 (also called PTPN11) were found to be required for cell survival upon hydroxyurea-induced replicative stress in HeLa cells. More detailed studies revealed that SHP2 was also important for the maintenance of the checkpoint after DNA damage induced by cisplatin or ionizing radiation in HeLa cells. Furthermore, SHP2 was activated after replicative stress and DNA damage. Although depletion of SHP2 resulted in a delay in cyclin E accumulation and an extension of G(1) phase, these cell cycle impairments were not responsible for the increase in apoptosis after DNA damage. Depletion of SHP2 impaired CHK1 activation, checkpoint-mediated cell cycle arrest, and DNA repair. These effects could be rescued with a shRNA-resistant SHP2. These results underscore the importance of protein phosphatases in checkpoint control and revealed a novel link between SHP2 and cell cycle checkpoints

Irreversible HER2 inhibitors overcome resistance to the RSL3 ferroptosis inducer in non-HER2 amplified luminal breast cancer

Abstract Ferroptosis, a form of programed cell death, can be promoted by inhibitors of the xCT transporter (erastin) or GPX4 (RSL3). We found that GPX4, but not the xCT transporter, is selectively elevated in luminal breast cancer. Consistent with this observation, the majority of luminal breast cancer cell lines are exquisitely sensitive to RSL3 with limited sensitivity to erastin. In RSL3-resistant, but not sensitive, luminal breast cancer cell lines, RSL3 induces HER2 pathway activation. Irreversible HER2 inhibitors including neratinib reversed RSL3 resistance in constitutively RSL3-resistant cell lines. Combination treatment with RSL3 and neratinib increases ferroptosis through mitochondrial iron-dependent reactive oxygen species production and lipid peroxidation. RSL3 also activated replication stress and concomitant S phase and G2/M blockade leading to sensitivity to targeting the DNA damage checkpoint. Together, our data support the exploration of RSL3 combined with irreversible HER2 inhibitors in clinical trials

SHP2 is involved in preventing mitotic entry after DNA damage.

<p>(<b>A</b>) SHP2 is involved in maintaining the G₂ DNA damage checkpoint. HeLa cells (expressing histone H2B–GFP) transfected with either control or SHP2 siRNA were irradiated with 10 Gy of IR. The time of mitotic entry of individual cell was tracked with time-lapse microscopy (<i>n</i> = 43–49). The data of the individual cells can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049943#pone.0049943.s007" target="_blank">Fig. S7A</a>. (<b>B</b>) SHP2 is involved in maintaining the G₂ DNA damage checkpoint. Cells were treated as in panel B and harvested at different time points. Lysates were prepared and analyzed with immunoblotting to confirm the knockdown of SHP2. The phosphorylation of histone H3^Ser10 indicated that cells transfected with siSHP2 were able to enter mitosis after IR treatment. (<b>C</b>) SHP2 is phosphorylated after DNA damage. HeLa cells were irradiated with 10 Gy of IR and harvested at the indicated time points. The cell pellets were directly boiled in sample buffer and analyzed with immunoblotting. (<b>D</b>) IR-induced CHK1 activation is impaired in SHP2-depleted cells. Control, siSHP2(a)-, or siSHP2(b)-transfected cells were treated with 10 Gy of IR and harvested at the indicated time points. Lysates were prepared and analyzed with immunoblotting.</p

SHP2 is activated by multiple agents that induce DNA replicative stress.

<p>(<b>A</b>) SHP2^Tyr542 is phosphorylated after replication stress. HeLa cells were incubated with HU for 64 h. The cells were harvested and immediately boiled in SDS sample buffer to preserve SHP2 phosphorylation. Phosphorylated SHP2^Tyr542 and total SHP2 were detected with immunoblotting. Uniform loading of lysates was assessed by actin analysis. (<b>B</b>) SHP2 is activated by a variety of stresses. HeLa cells were treated thymidine (THY), aphidicolin (APH), HU, CIS (2 µg/ml), camptothecin (CAM), Adriamycin (ADR), or etoposide (ETP). The cells were harvested after 48 h. Phosphorylated SHP2^Tyr542 and total SHP2 were detected with immunoblotting. Uniform loading of lysates was assessed by actin analysis.</p