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Background and Purpose: Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. Experimental Approach: The mechanisms of action and therapeutic effects of selective pan‐FGFR inhibitors (pan‐FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine‐sensitive to highly gemcitabine‐resistant (GemR). Gain‐/loss‐of‐function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. Results: The pan‐FGFRi NVP‐BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival‐ and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard‐of‐care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss‐of‐function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi‐mediated inhibition of proliferation and migration. Single‐agent FGFRi suppressed the viability and growth of multiple patient‐derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1‐dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. Conclusions and Implications: Single‐agent FGFR inhibitors mediate selective, molecularly‐targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low‐to‐moderate levels of FGFR1. [ABSTRACT FROM AUTHOR]

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent., Introduction Polyamines are small polycationic molecules that have been implicated in virtually every aspect of eukaryotic cells, including DNA replication, transcription, translation, and proliferation (1-3). Polyamine levels are increased in [...]

Malignant melanoma claims more lives than any other skin malignancy. While primary melanomas are usually cured via surgical excision, the metastatic form of the disease portents a poor prognosis. Decades of intense research has yielded an extensive armamentarium of anti-melanoma therapies, ranging from genotoxic chemo- and radiotherapies to targeted interventions in specific signaling pathways and immune functions. Unfortunately, even the most up-to-date embodiments of these therapies are not curative for the majority of metastatic melanoma patients, and the need to improve their efficacy is widely recognized. Here, we review the reports that implicate p21-regulated kinase 1 (PAK1) and PAK1-related pathways in the response of melanoma to various therapeutic modalities. Ample data suggest that PAK1 may decrease cell sensitivity to programmed cell death, provide additional stimulation to growth-promoting molecular pathways, and contribute to the creation of an immunosuppressive tumor microenvironment. Accordingly, there is mounting evidence that the concomitant inhibition of PAK1 enhances the potency of various anti-melanoma regimens. Overall, the available information suggests that a safe and effective inhibition of PAK1-dependent molecular processes would enhance the potency of the currently available anti-melanoma treatments, although considerable challenges in implementing such strategies still exist. [ABSTRACT FROM AUTHOR]

Although melanoma accounts for only 5.3% of skin cancer, it results in >75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ. [ABSTRACT FROM AUTHOR]

Author(s): Ilya Shamovsky [1, 2]; Maxim Ivannikov [1]; Eugene S. Kandel [3]; David Gershon (corresponding author) [2, 4]; Evgeny Nudler (corresponding author) [1] The heat-shock transcription factor 1 (HSF1) has [...]

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-[kappa]B- dependent signaling in human cells. Two validated targets are the re/A gene, which codes for the NF-[kappa]B p65 subunit, and the NF-[kappa]B regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-[kappa]B-dependent secretion of factors that activate NF-[kappa]B through cell-surface receptors, establishing an autocrine loop. Act1 | forward genetics | p65 RelA

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury. [ABSTRACT FROM AUTHOR]

MicroRNAs are short RNA molecules that regulate function and stability of a large subset of eukaryotic mRNAs. In the main pathway of microRNA biogenesis, a short “hairpin” is excised from a primary transcript by ribonuclease DROSHA, followed by additional nucleolytic processing by DICER and inclusion of the mature microRNA into the RNA-induced silencing complex. We report that a microRNA-like molecule is encoded by human DROSHA gene within a predicted stem-loop element of the respective transcript. This putative mature microRNA is complementary to DROSHA transcript variant 1 and can attenuate expression of the corresponding protein. The findings suggest a possibility for a negative feedback loop, wherein DROSHA processes its own transcript and produces an inhibitor of its own biosynthesis. [ABSTRACT FROM PUBLISHER]

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 1990's 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. [ABSTRACT FROM AUTHOR]

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 NFκB, 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. [ABSTRACT FROM AUTHOR]

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance. [ABSTRACT FROM AUTHOR]

Background: Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell. Methodology/Principal Findings: We tested the ability of XMRV to complement replication-deficient MLV vectors upon coinfection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 104 infectious vector particles per milliliter of culture medium. Conclusions/Significance: The possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1-/- fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin β1 blocking antibodies, treatment with β1-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1-/- fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin α5β1. [ABSTRACT FROM AUTHOR]

p21 is a potent inhibitor of cyclin-dependent kinases capable of arresting cell cycle progression. p21 is primarily regulated at the transcriptional level by several transcription factors, including p53. Previously, we reported that certain members of the E2F family of transcription factors may activate p21 transcription via a p53-independent mechanism. To further elucidate the consequences of E2F-1-regulated induction of p21, we developed cell lines with a tamoxifen-dependent form of E2F-1. We confirmed direct interaction of E2F-1 with the proximal region of the p21 promoter. Interestingly, elevated E2F-1 activity was sufficient to arrest a substantial subset of cells in S phase and this effect was correlated to and dependent on the induction of p21 protein. Since E2F proteins control genes required for cell cycle progression and are activated by various oncogenic events, we believe that the p21-dependent arrest described in this report represents an additional mechanism that guards against unrestricted cell proliferation. [ABSTRACT FROM AUTHOR]

Transfection of retrovirus packaging cells with linear DNA from a retroviral vector missing the 3′ long terminal repeat (3′ LTR) results in production of infectious virus. Analysis of the newly formed proviruses indicates that restoration of the 3′ LTR sequences necessary for reverse transcription and integration occurred due to end-to-end template switching by mammalian RNA polymerase II (RNAP II) in the packaging cells. These observations argue that RNAP II can utilize double-strand breaks and gaps in DNA to generate “recombinant” transcripts in vivo and suggest a mechanism for mutation and recombination of retroviruses. [Copyright &y& Elsevier]

Summary Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress. Graphical Abstract Highlights • XBP1s transcriptionally activates KLF9 under conditions of high ER stress • KLF9 transcriptionally activates TMEM38B and ITPR1 to control ER calcium release • KLF9 further increases ER stress via facilitating calcium release from ER • Klf9 deficiency reduces tunicamycin-induced ER stress in mouse liver The transcription factor XBP1s plays a central role in suppression of endoplasmic reticulum (ER) stress through direct activation of multiple genes controlling protein refolding. Fink et al. report that elevation of ER stress above a certain threshold triggers an XBP1s-dependent transcriptional program, leading to exacerbation of ER stress and cell death. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Identifies new genes controlling cell response to tumor necrosis factor (TNF) using the genetic suppressor element (GSE) methodology. Screening of a retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts for GSE capable of protecting NIH3T3 cells from TNF-induced apoptosis; Dependence of the GSE-mediated activation of the NF-kappa-B on the presence of serum.