Eugene S. Kandel

miRNAs: Little known mediators of oncogenesis

Cancer progression is mediated by overexpression of oncogenes and downregulation or loss of tumor suppressors. Proteins, which were traditionally categorized into these groups, have been recently joined by a species of RNA molecules known as microRNAs (miRNAs). miRNAs belong to a class of approximately 22-nt-long non-coding RNAs found in eukaryotes that hinder gene expression by inducing degradation or inhibiting translation of select mRNAs. A growing number of miRNAs have been implicated in promoting or suppressing tumorigenesis in a variety of tissues. The supporting evidence ranges from suggestive expression profiling data to direct functional validation using methods of forward and reverse genetics. We discuss the nature of published results, as well as the merits and pitfalls of various approaches aimed at identification of cancer-related miRNAs and their mRNA targets.

PAK1 as a therapeutic target

Importance of the field: P21-activated kinases (PAKs) are involved in multiple signal transduction pathways in mammalian cells. PAKs, and PAK1 in particular, play a role in such disorders as cancer, mental retardation and allergy. Cell motility, survival and proliferation, the organization and function of cytoskeleton and extracellular matrix, transcription and translation are among the processes affected by PAK1. Areas covered in this review: We discuss the mechanisms that control PAK1 activity, its involvement in physiological and pathophysiological processes, the benefits and the drawbacks of the current tools to regulate PAK1 activity, the evidence that suggests PAK1 as a therapeutic target and the likely directions of future research. What the reader will gain: The reader will gain a better knowledge and understanding of the areas described above. Take home message: PAK1 is a promising therapeutic target in cancer and allergen-induced disorders. Its suitability as a target in vascular, neurological and infectious diseases remains ambiguous. Further advancement of this field requires progress on such issues as the development of specific and clinically acceptable inhibitors, the choice between targeting one or multiple PAK isoforms, elucidation of the individual roles of PAK1 targets and the mechanisms that may circumvent inhibition of PAK1.

Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10−6 or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFκB. High levels of FBXL11 block the ability of NFκB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFκB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFκB-dependent signaling.

Nrf2 Amplifies Oxidative Stress via Induction of Klf9

Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury.

The role of PAK-1 in activation of MAP kinase cascade and oncogenic transformation by Akt

The activity of protein kinase B, also known as Akt, is commonly elevated in human malignancies and plays a crucial role in oncogenic transformation. The relationship between Akt and the mitogen-activated protein kinase cascade, which is also frequently associated with oncogenesis, remains controversial. We report here examples of cooperation between Akt and cRaf in oncogenic transformation, which was accompanied by elevated activity of extracellular signal-regulated mitogen-activated protein kinases. The effect of Akt on extracellular signal-regulated kinases depended on the status of p21-activated kinase (PAK). Importantly, disruption of the function of PAK not only uncoupled the activation of Akt from that of extracellular signal-regulated kinases, but also greatly reduced the capacity of Akt to act as a transforming oncogene. For the malignancies with hyperactive Akt, our observations support the role for PAK-1 as a potential target for therapeutic intervention.

Absence of XMRV and closely related viruses in primary prostate cancer tissues used to derive the XMRV-infected cell line 22Rv1

The 22Rv1 cell line is widely used for prostate cancer research and other studies throughout the world. These cells were established from a human prostate tumor, CWR22, that was serially passaged in nude mice and selected for androgen independence. The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990s when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus. The primary tumor tissues lacked mouse DNA as determined by PCR for intracisternal A type particle DNA, thus avoiding one of the limitations of studying xenografts. We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells.

A GOG 210 aCGH study of gain at 1q23 in endometrioid endometrial cancer in the context of racial disparity and outcome

The goal of this study was to identify recurrent regions of genomic gain or loss in endometrial cancer of the endometrioid type in the context of racial disparities in mortality for this disease. Array comparative genomic hybridization (aCGH) analysis was performed on 80 frozen primary tumors from the Gynecologic Oncology Group (GOG)‐210 bank using the RPCI 19K BAC arrays. The 80 patients included 20 African American (AA) Stage I, 20 White (W) Stage I, 20 African American (AA) Stage IIIC/IV, and 20 White (W) Stage IIIC/IV. A separate subset of 220 endometrial cancers with outcome data was used for validation. A 1.6‐Mbp region of gain at 1q23 was identified by aCGH in all AA patients and high grade W patients, but not W low grade patients. In the validation arm of 220 patients copy number gain at this region was validated using FISH and locus specific BACs. The number of AA patients in the validation arm was too small to confirm the aCGH association with racial disparity. Kaplan‐Meier curves for survival showed a significant difference for gain at 1q23 versus no gain (log rank P = 0.0014). When subdivided into various groups of risk by stage and grade the survival curves showed a decreased survival for high grade and/or stage tumors, but not for low grade and/or stage endometrioid tumors. Univariate analyses for gain at 1q23 showed a significant association (P = 0.009) with survival. Multivariate analysis for gain at 1q23 did not show a significant association with survival (P = 0.14).

Long-Distance Effects of Insertional Mutagenesis

Background Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance. Methodology/Principal Findings We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected. Conclusions/Significance We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.

P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells

BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti‐cancer agents, BRAF‐inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi‐insensitivity in many BRAF‐mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi‐resistance. We explored the role of p21‐activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi‐resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto‐oncogenes increased BRAFi‐tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK‐ and BRAF‐inhibitors synergistically affected intrinsically BRAFi‐resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well‐known resistance mechanisms.

Transposon-based mutagenesis identifies short RIP1 as an activator of NFκB

We used a vector based on the Sleeping Beauty transposon to search for constitutive activators of NF-κB in cultured cells. Dominant mutations were produced by random insertion of a tetracycline-regulated promoter, which provided robust and exceptionally well-regulatedexpression of downstream genes. The ability to regulate the mutant phenotype was used to attribute the latter to the insertional event. In one such mutant, the promoter was inserted in the middle of the gene encoding receptor-interacting protein kinase 1 (RIP1). The protein encoded by the hybrid transcript lacks the putative kinase domain of RIP1, but potently stimulates NF-κB, AP-1 and Ets-1 activity. Similarly to TNFα treatment, expression of the short RIP1 was toxic to cells that failed to up-regulate NF-κB. The effects of short RIP1 did not require endogenous RIP1 or cytokine treatment and coincided with reduced responsiveness to TNFα. Additional evidenceindicates that a similar short RIP1 could be produced naturally from the ripk1 locus. Interestingly, elevated expression of short RIP1 resulted in the loss of full length RIP1 from cells, pointing to a novel mechanism through which the abundance of RIP1 and the status of the related signaling cascades may be regulated.