shRNA libraries and their use in cancer genetics

Bernards, René; Brummelkamp, Thijn R.; Beijersbergen, Roderick L.

doi:10.1038/nmeth921

Cited by 121 publications

(86 citation statements)

References 27 publications

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“…To gain insights into the regulation of cell proliferation by Ras proteins beyond the Raf/Mek/Erk cascade and to identify key elements that connect Ras signaling with the cell cycle machinery, we submitted mouse embryonic fibroblasts (MEFs) lacking all of the Ras proteins (Rasless cells) to an unbiased shRNA library "barcode" screen (10,11) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

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Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

Drosten

Sum

Lechuga

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/ p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N-and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism.T he RAS genes have been extensively studied due to their key role in mediating mitogenic signaling as well as their high prevalence in human cancers, including those cancers with poor survival rates, such as lung adenocarcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma (1, 2). However, the mechanisms by which Ras proteins mediate mitogenic signaling in either normal or tumor cells remain obscure, especially beyond activation of the Raf/Mek/Erk cascade. Recent genetic studies have underscored the relevance of Ras proteins in cellular homeostasis by demonstrating that cells lacking the three Ras loci, H-Ras, N-Ras, and K-Ras (Rasless cells), are completely unable to proliferate (3, 4). Indeed, systemic ablation of these loci in adult mice causes rapid deterioration of multiple tissues, leading to their death in a few days.GTP-loaded Ras proteins promote activation of various downstream signal transduction pathways, mainly the Raf/Mek/Erk kinase cascade, the PI3K/Akt route, and the Ral guanine dissociation stimulator (RalGDS) pathway (1). Activation of other pathways, such as those pathways driven by the Rac family of small G proteins and phospholipase C, has also been illustrated (1). However, genetic interrogation of the pathways essential for cell proliferation has illustrated that only constitutive activation of the Raf, Mek, or Erk kinase can bypass the requirement for Ras proteins to sustain cell division, at least in vitro (3, 4). Indeed, constitutive activation of the PI3K/Akt and RalGDS pathways was incapable of inducing cell proliferation in the absence of Ras proteins. In agreement wit...

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Section: Resultsmentioning

confidence: 99%

“…1A). K-Raslox cells were transfected with the shRNA library and scored for cells capable of proliferating in the absence of Ras proteins (10,11) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

Drosten

Sum

Lechuga

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…With the advent of RNA interference technology, it has become possible to systematically determine the functional consequence of gene suppression in cancer cell lines (Downward, 2004;Bernards et al, 2006;Iorns et al, 2007). Isogenic paired cancer cell lines that differ by a single oncogenic lesion can be used to identify potential targets for selectively killing tumour cells.…”

Section: Introductionmentioning

confidence: 99%

Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells

et al. 2010

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Activating mutations in the KRAS gene are among the most prevalent genetic changes in human cancers. To identify synthetic lethal interactions in cancer cells harbouring mutant KRAS, we performed a large-scale screen in isogenic paired colon cancer cell lines that differ by a single allele of mutant KRAS using an inducible short hairpin RNA interference library. Snail2, a zinc finger transcriptional repressor encoded by the SNAI2 gene, was found to be selectively required for the long-term survival of cancer cells with mutant KRAS that have undergone epithelial-mesenchymal transition (EMT), a transdifferentiation event that is frequently seen in advanced tumours and is promoted by RAS activation. Snail2 expression is regulated by the RAS pathway and is required for EMT. Our findings support Snail2 as a possible target for the treatment of the broad spectrum of human cancers of epithelial origin with mutant RAS that have undergone EMT and are characterized by a high degree of chemoresistance and radioresistance.

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“…In addition, to obtain better understanding of the role of gankyrin in ESCC we used a plasmid vector short-hairpin RNA (shRNA) expression system in vitro and in vivo, that provides a powerful tool used for inducing the loss of function phenotypes by posttranscriptional gene silencing. 13,14 Material and methods …”

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confidence: 99%

Gankyrin oncoprotein overexpression as a critical factor for tumor growth in human esophageal squamous cell carcinoma and its clinical significance

Ortiz

Ito

Tanaka

et al. 2007

Intl Journal of Cancer

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To elucidate the possible involvement of gankyrin in ESCC progression and the effect of its down-regulation in ESCC, we investigated the expression of gankyrin in ESCC tissues comparing it with the corresponding normal esophageal epithelia and tested a short-hairpin RNA (shRNA) expression vector for gankyrin in ESCC cell lines. Gankyrin protein expression in 11 ESCC cell lines (KYSE series) was examined by RT-PCR and western blot. The expression of gankyrin mRNA in 30 ESCC tissues was compared with the corresponding normal epithelia by Real-time PCR. Expression of gankyrin protein was immunohistochemically analyzed in the ESCC of 103 patients. A gankyrin-shRNA vector was stably transfected into KYSE 170 cells to assess the role of gankyrin in cell motility, invasion and proliferation in vitro and tumor formation in vivo. Gankyrin expression increased in all 11 ESCC cell lines. Real-time PCR revealed that gankyrin expression was higher in the cancerous tissue for all 30 patients. In immunohistochemistry, gankyrin overexpression was correlated with lower survival rate (p 5 0.0001), extent of the primary tumor, lymph node metastasis, distant lymph node metastasis and stage (p 5 0.0072, p 5 0.0004, p 5 0.0172 and p 5 0.0002, respectively). A shRNA vector against gankyrin repressed growth, cell motility, invasiveness in vitro and tumor formation in vivo. Gankyrin overexpression is associated with poor prognosis. It may play an important role in ESCC tumor progression and could be a potentially important therapeutic gene target in ESCC. ' 2007 Wiley-Liss, Inc.Key words: gankyrin; oncogene; esophageal cancer; clinical significance; novel; cell cycle Despite considerable advances in surgical techniques, perioperative care and neoadjuvant chemoradiotherapy, esophageal squamous cell carcinoma (ESCC) still remains one of the most lethal cancers and the seventh leading cause of cancer death worldwide. 1,2 Gene study is widely performed in ESCC 3-5 and new oncogenes associated with the progression of ESCC have been identified and targeted to improve the survival of patients with this type of refractory cancer.Gankyrin is a novel oncoprotein with 7 ankyrin repeats. 6-8 Overexpression of gankyrin increases the phosphorylation and degradation of retinoblastoma (RB1) by its RB1-binding motif, suggesting that it promotes tumorigenicity and cancer cell proliferation. 9 Furthermore, binding to CDK4 and counteracting the inhibitory function of the INK4 proteins, including tumor suppressors p16 INK4A and p18 INK4B , gankyrin acts as an accelerator for cell cycle progression. 9-11 Gankyrin also binds to MDM2, facilitating MDM2-p53 interaction and increasing its polyubiquitilation and degradation. 12 These findings suggest the importance of gankyrin in the cell cycle control and tumorigenesis. However, the physiological and physiopathological role of gankyrin in the cell cycle progression of normal cells remains unknown.Overexpression of gankyrin has been found ubiquitously in hepatocellular carcinoma tissues, liver regeneration and f...

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shRNA libraries and their use in cancer genetics

Cited by 121 publications

References 27 publications

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells

Gankyrin oncoprotein overexpression as a critical factor for tumor growth in human esophageal squamous cell carcinoma and its clinical significance

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