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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., (© 2023 British Pharmacological Society.)

Aims: Pancreatic ductal adenocarcinoma (PDAC) is often intrinsically-resistant to standard-of-care chemotherapies such as gemcitabine. Acquired gemcitabine resistance (GemR) can arise from treatment of initially-sensitive tumors, and chemotherapy can increase tumor aggressiveness. We investigated the molecular mechanisms of chemoresistance and chemotherapy-driven tumor aggressiveness, which are understood incompletely., Methods: Differential proteomic analysis was employed to investigate chemotherapy-driven chemoresistance drivers and responses of PDAC cells and patient-derived tumor xenografts (PDX) having different chemosensitivities. We also investigated the prognostic value of FGFR1 expression in the efficacy of selective pan-FGFR inhibitor (FGFRi)-gemcitabine combinations., Results: Quantitative proteomic analysis of a highly-GemR cell line revealed fibroblast growth factor receptor 1 (FGFR1) as the highest-expressed receptor tyrosine kinase. FGFR1 knockdown or FGFRi co-treatment enhanced gemcitabine efficacy and decreased GemR marker expression, implicating FGFR1 in augmentation of GemR. FGFRi treatment reduced PDX tumor progression and prolonged survival significantly, even in highly-resistant tumors in which neither single-agent showed efficacy. Gemcitabine exacerbated aggressiveness of highly-GemR tumors, based upon proliferation and metastatic markers. Combining FGFRi with gemcitabine or gemcitabine+nab-paclitaxel reversed tumor aggressiveness and progression, and prolonged survival significantly. In multiple PDAC PDXs, FGFR1 expression correlated with intrinsic tumor gemcitabine sensitivity., Conclusion: FGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse., Competing Interests: Declaration of Competing Interest Authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

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.

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells., (© 2021. The Author(s).)

The activation of oncogenic mitogen-activated protein kinase cascade via mutations in BRAF is often observed in human melanomas. Targeted inhibitors of BRAF (BRAFi), alone or as a part of a combination therapy, offer a significant benefit to such patients. Unfortunately, some cases are initially nonresponsive to these drugs, while others become refractory in the course of treatment, underscoring the need to understand and mitigate the underlying resistance mechanisms. We report that interference with polo-like kinase 3 (PLK3) reduces the tolerance of BRAF-mutant melanoma cells to BRAFi, while increased PLK3 expression has the opposite effect. Accordingly, PLK3 expression correlates with tolerance to BRAFi in a panel of BRAF-mutant cell lines and is elevated in a subset of recurring BRAFi-resistant melanomas. In PLK3-expressing cells, R406, a kinase inhibitor whose targets include PLK3, recapitulates the sensitizing effects of genetic PLK3 inhibitors. The findings support a role for PLK3 as a predictor of BRAFi efficacy and suggest suppression of PLK3 as a way to improve the efficacy of targeted therapy., (© 2019 Wiley Periodicals, Inc.)

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: Braf CA mice, where Braf V600E causes premalignant melanocytic hyperplasia, and Braf CA /Pten -/- mice, where Braf V600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in Braf CA mice but did not affect formation and growth of Braf CA /Pten -/- primary melanomas. It also, as expected, promoted Braf CA /Pten -/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in Braf CA and Braf CA /Pten -/- melanocytic cells. Mechanistically, we demonstrated that BRAF V600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAF V600E . PTEN depletion in BRAF V600E -melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAF V600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

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.

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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAF V600E -dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.

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.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

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., (© 2017 Wiley Periodicals, Inc.)

It is increasingly clear that the biological functions of a transcription factor cannot be fully understood solely on the basis of protein-coding genes that fall under its control. Many transcription factors regulate expression of miRNAs, which affect the cell by modulating translation and stability of mRNAs. The identities and the roles of NF-κB-regulated miRNAs have been attracting research interest for a long time. We revisited this issue in a system with controlled expression of one of the key regulators of NF-κB, RIPK1. Several regulated miRNAs were identified, including miR-146a, miR-215 and miR-497. The miRNAs were also inducible by IL-1β, but not when NF-κB activity was repressed by mutant IκBα. The presence of a miR-497 site was predicted in the 3'-UTR of IKBKB gene, which encodes IKKβ. Using appropriately engineered reporters, we confirmed that this site can be a target of suppressive action of miR-497. Our findings suggest that NF-κB controls expression of a miRNA, which may reduce production of IKKβ. Considering the role of IKKβ in the canonical pathway of NF-κB activation, our observations may indicate a new mechanism that modulates the magnitude of such activation, as well as the propensity of a cell to engage canonical vs. non-canonical pathways.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6-22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy.

ETV1 (ETS variant 1) is a transcription factor from the ETS family and an oncogene in several types of human malignancies. Paradoxically, a predicted inactivating mutation in ETV1 was previously found in a clone of HT1080 cells with reduced activity of p53. We report that elevated expression of ETV1 makes p53-null tumor cells hypersensitive to restoration of said tumor suppressor. Furthermore, elevated levels of either wild-type ETV1 or its truncated derivative, dETV1, which mimics the product of an oncogenic rearrangement in certain tumors, results in increased expression of mRNA for p14ARF, a known activator of p53. Accordingly, expression of a luciferase reporter, which is driven by a putative ARF promoter, was elevated by concomitant expression of either ETV1 or dETV1. Our observations point to yet another example of a tumor suppressor gene being activated by a potentially oncogenic signal. A better understanding of the mechanisms that allow a cell to bypass such safeguards is needed in order to predict and prevent the development of an oncogene-tolerant state during cancer evolution.

Phosphoinositol-3-kinase (PI3K) pathway is a crucial modulator of many physiological and pathophysiological phenomena, including aging, diabetes and cancer. Protein kinase Akt, a downstream effector of PI3K, controls a plethora of cellular functions, including gene transcription. A key mechanism connecting Akt activity to changes in gene expression is inhibitory phosphorylation of FOXO family of transcription factors. Accordingly, altered expression of FOXO targets may account for many biological consequences of PI3K/Akt signaling. While the previous efforts focused on FOXO-dependent regulation of protein-coding genes, non-coding RNA genes have emerged as equally important targets of many transcription factors. Therefore, we utilized a regulated form of FOXO1 to profile FOXO1-dependent changes in miRNA expression in human cells. Both microarray hybridization and next-generation sequencing revealed changes in the products of a miRNA cluster on X chromosome. Rapid induction of these miRNAs occurred independently of de novo protein synthesis. Furthermore, inhibition of PI3K in cancer cell lines caused derepression of these miRNAs, as would be expected for FOXO-regulated genes. Members of the major oncogenic cascades are significantly overrepresented among the predicted targets of the miRNAs, consistent with tumor-suppressive role of FOXO1. The discovered miRNAs represent new candidate mediators of FOXO1 functions and possible biomarkers of its activity.

While new drugs aimed at BRAF-mutated cancers are entering clinical practice, cells and tumors with activating Ras mutations are relatively resistant to those and quite a few other anti-cancer agents. This inspires the effort to reverse this resistance or to uncover new vulnerabilities in such resistant cancers. IPA3 has been originally identified as a small molecule inhibitor of p21-activated protein kinase 1 (PAK1), a candidate therapeutic target in human malignancies. We have tested a battery of melanoma and colon carcinoma cell lines that carry mutations in BRAF, NRAS and KRAS genes and have observed that those with NRAS and KRAS mutations are more sensitive to killing by IPA3. Genetic manipulations suggest that the differential response depends not just on these oncogenes, but also on additional events that were co-selected during tumor evolution. Furthermore, sublethal doses of IPA3 or ectopic expression of dominant-negative PAK1 sensitized Ras-mutated cells to GDC-0897 and AZD6244, which otherwise have reduced efficiency against cells with activated Ras. Dominant-negative PAK1 also reduced the growth of NRAS-mutated cells in confluent cultures, but, unlike IPA3, caused no significant toxicity. Although it remains to be proven that all the effects of IPA3 are exclusively due to inhibition of PAK1, our findings point to the existence of selective vulnerabilities, which are associated with Ras mutations and could be useful for better understanding and treatment of a large subset of tumors.

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.

The prevalence of xenotropic murine leukemia virus-related virus (XMRV) in human population and its involvement in prostate cancer are subjects of ongoing research and debate. 22Rv1, which is a human cell line that serves as a common model of androgen-independent prostate cancer, was recently reported to carry infectious copies of XMRV. 22Rv1 was derived from a prostate cancer xenograft CWR22 that was serially passaged in immunodeficient mice. Based on the analysis of the DNA from CWR22 and 22Rv1, we present evidence against the presence of XMRV in CWR22 and, by inference, the tumor, from which CWR22 and 22Rv1 were established. While the presence of XMRV in 22Rv1 is likely to be an artifact, it may be a significant factor in determining the biological properties of this cell line. This consideration warrants additional caution for the interpretation of the relevance of the studies, which utilize this popular cell line as a model. It also invites a closer look at the sources of viral contamination in xenografts and cultured cells, as well as in the experiments that allege the presence of this virus in human cells and populations.

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.

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.

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 NFkappaB. High levels of FBXL11 block the ability of NFkappaB 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 NFkappaB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFkappaB-dependent signaling.

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 co-infection 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 10(4) 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.

We used a vector based on the Sleeping Beauty transposon to search for constitutive activators of NFkappaB in cultured cells. Dominant mutations were produced by random insertion of a tetracycline-regulated promoter, which provided robust and exceptionally well-regulated expression 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 NFkappaB, AP-1 and Ets-1 activity. Similarly to TNFalpha treatment, expression of the short RIP1 was toxic to cells that failed to upregulate NFkappaB. The effects of short RIP1 did not require endogenous RIP1 or cytokine treatment and coincided with reduced responsiveness to TNFalpha. Additional evidence indicates 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.

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.

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 beta(1) blocking antibodies, treatment with beta(1)-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 alpha(5)beta(1).

The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.

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-kappaB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-kappaB p65 subunit, and the NF-kappaB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-kappaB-dependent secretion of factors that activate NF-kappaB through cell-surface receptors, establishing an autocrine loop.

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.

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.

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.

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.