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Background Long noncoding RNAs (lncRNAs) are an emerging class of relatively underexplored oncogenic molecules with biological and clinical significance. Current inadequacies for stratifying patients with aggressive disease presents a strong rationale to systematically identify lncRNAs as clinical predictors in localized prostate cancer. Objective To identify RNA biomarkers associated with aggressive prostate cancer. Design, setting, and participants Radical prostatectomy microarray and clinical data was obtained from 910 patients in three published institutional cohorts: Mayo Clinic I ( N =545, median follow-up 13.8 yr), Mayo Clinic II ( N =235, median follow-up 6.7 yr), and Thomas Jefferson University ( N =130, median follow-up 9.6 yr). Outcome measurements and statistical analysis The primary clinical endpoint was distant metastasis-free survival. Secondary endpoints include prostate cancer-specific survival and overall survival. Univariate and multivariate Cox regression were used to evaluate the association of lncRNA expression and these endpoints. Results and limitations An integrative analysis revealed Prostate Cancer Associated Transcript-14 ( PCAT-14 ) as the most prevalent lncRNA that is aberrantly expressed in prostate cancer patients. Down-regulation of PCAT-14 expression significantly associated with Gleason score and a greater probability of metastatic progression, overall survival, and prostate cancer-specific mortality across multiple independent datasets and ethnicities. Low PCAT-14 expression was implicated with genes involved in biological processes promoting aggressive disease. In-vitro analysis confirmed that low PCAT-14 expression increased migration while overexpressing PCAT-14 reduced cellular growth, migration, and invasion. Conclusions We discovered that androgen-regulated PCAT-14 is overexpressed in prostate cancer, suppresses invasive phenotypes, and lower expression is significantly prognostic for multiple clinical endpoints supporting its significance for predicting metastatic disease that could be used to improve patient management. Patient summary We discovered that aberrant prostate cancer associated transcript-14 expression during prostate cancer progression is prevalent across cancer patients. Prostate cancer associated transcript-14 is also prognostic for metastatic disease and survival highlighting its importance for stratifying patients that could benefit from treatment intensification.

// Zhi Hong Lu 1, 2 , Sergey Kaliberov 2, 3, 4 , Rebecca E. Sohn 1, 2 , Lyudmila Kaliberova 2, 3, 4 , Yingqiu Du 1, 2 , Julie L. Prior 5 , Daniel J. Leib 6 , Anne Chauchereau 7 , Jennifer K. Sehn 2, 8 , David T. Curiel 2, 3, 4 , Jeffrey M. Arbeit 1, 2 1 Urology Division and Department of Surgery, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 2 Siteman Cancer Center, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 3 Biologic Therapeutics Center, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 4 Department of Radiation Oncology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 5 Radiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 6 Department of Orthopedic Surgery, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA 7 Prostate Cancer Group, INSERM U981, Gustave Roussy, Villejuif, F-94805, France 8 Department of Anatomic and Molecular Pathology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA Correspondence to: Jeffrey M. Arbeit, email: arbeitj@wudosis.wustl.edu David T. Curiel, email: dcuriel@radonc.wustl.edu Keywords: prostate, metastasis, endothelial, niche, adenovirus Received: August 12, 2016 Accepted: December 26, 2016 Published: January 17, 2017 ABSTRACT While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.

Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter were characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.

Vascular disrupting agents (VDA) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow–derived circulating endothelial precursor (CEP) cells could promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here, we show that following treatment of mice with OXi-4503, a second-generation potent prodrug derivative of combretastatin-A4 phosphate, rapid increases in circulating plasma vascular endothelial growth factor, stromal derived factor-1 (SDF-1), and granulocyte colony-stimulating factor (G-CSF) levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R−/− mice were treated with OXi-4503. We found that as opposed to wild-type controls, G-CSF-R−/− mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared with tumors treated in wild-type mice. Evidence for rapid elevations in circulating plasma G-CSF, vascular endothelial growth factor, and SDF-1 were also observed in patients with VDA (combretastatin-A4 phosphate)-treated cancer. These results highlight the possible effect of drug-induced G-CSF on tumor regrowth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target. [Cancer Res 2009;69(19):7524–8]

The phosphoinositide 3-kinase (PI3K) pathway regulates mammalian cell growth, survival, and motility and plays a major pathogenetic role in human prostate cancer (PCa). However, the oncogenic contributions downstream of the PI3K pathway made by mammalian target of rapamycin complex 1 (mTORC1)–mediated cell growth signal transduction in PCa have yet to be elucidated in detail. Here, we engineered constitutive mTORC1 activation in prostate epithelium by a conditional genetic deletion of tuberous sclerosis complex 1 (Tsc1), a potent negative regulator of mTORC1 signaling. Epithelial inactivation was not immediately tumorigenic, but Tsc1-deficient mice developed prostatic intraepithelial neoplasia (mPIN) in lateral and anterior prostates by 6 months of age, with increasing disease penetrance over time. Lateral prostate lesions in 16- to 22-month-old mutant mice progressed to two types of more advanced lesions, adenomatous gland forming lesion (Type 1) and atypical glands embedded in massively expanded reactive stroma (Type 2). Both Type 1 and Type 2 lesions contained multiple foci of microinvasive carcinoma. Epithelial neoplastic and atypical stromal lesions persisted despite 4 weeks of RAD001 chemotherapy. Rapalogue resistance was not due to AKT or extracellular signal-regulated kinase 1/2 activation. Expression of the homeobox gene Nkx3.1 was lost in Tsc1-deficient mPIN, and it cooperated with TSC1 loss in mPIN initiation in doubly mutant Tsc1:Nkx3.1 prostatic epithelial knockout mice. Thus, TSC1 inactivation distal to PI3K and AKT activation is sufficient to activate a molecular signaling cascade producing prostatic neoplasia and focal carcinogenesis. Cancer Res; 70(21); 8937–47. ©2010 AACR.

Noninvasive detection of dysplasia provides a potential platform for monitoring the efficacy of chemopreventive therapy of premalignancy, imaging the tissue compartments comprising dysplasia: epithelium, microvasculature, and stromal inflammatory cells. Here, using respiratory-gated magnetic resonance imaging (MRI), the anatomy of premalignant and malignant stages of cervical carcinogenesis in estrogen-treated K14-HPV16 transgenic mice was noninvasively defined. Dynamic contrast enhanced (DCE)-MRI was used to quantify leakage across premalignant dysplastic microvasculature. Vascular permeability as measured by DCE-MRI, Ktrans, was similar in transgenic (0.053 ± 0.020 min−1; n = 32 mice) and nontransgenic (0.056 ± 0.029 min−1; n = 17 mice) animals despite a 2-fold increase in microvascular area in the former compared with the latter. DCE-MRI did detect a significant decrease in vascular permeability accompanying diminution of dysplastic microvasculature by the antiangiogenic agent, vascular endothelial growth factor Trap (Ktrans = 0.052 ± 0.013 min−1 pretreatment; n = 6 mice versus Ktrans = 0.019 ± 0.008 min−1 post-treatment; n = 5 mice). Thus, we determined that the threshold of microvessel leakage associated with cervical dysplasia was

The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.

Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression.In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice.Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ.In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities.

Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques.This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

Hypoxia-inducible factor-1alpha (HIF-1alpha) transactivates genes required for energy metabolism and tissue perfusion and is necessary for embryonic development and tumor explant growth. HIF-1alpha is overexpressed during carcinogenesis, myocardial infarction, and wound healing; however, the biological consequences of HIF-1alpha overexpression are unknown. Here, transgenic mice expressing constitutively active HIF-1alpha in epidermis displayed a 66% increase in dermal capillaries, a 13-fold elevation of total vascular endothelial growth factor (VEGF) expression, and a six- to ninefold induction of each VEGF isoform. Despite marked induction of hypervascularity, HIF-1alpha did not induce edema, inflammation, or vascular leakage, phenotypes developing in transgenic mice overexpressing VEGF cDNA in skin. Remarkably, blood vessel leakage resistance induced by HIF-1alpha overexpression was not caused by up-regulation of angiopoietin-1 or angiopoietin-2. Hypervascularity induced by HIF-1alpha could improve therapy of tissue ischemia.

The cytokine interferon-beta is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-beta by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-beta protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-beta whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-beta protein. Neutralizing interferon-beta in the culture medium inhibited differentiation, but the addition of exogenous interferon-beta did not stimulate differentiation. These data indicate that interferon-beta is produced by growth-arrested, terminally differentiated keratinocytes.

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation., Author Summary The human bone tissue is of considerable regenerative capacity as reflected in bone remodeling and in fracture healing. However, bone tissue regeneration deteriorates with age, and tremendous unmet medical needs exist for safe and effective strategies to stimulate bone formation in older individuals commonly inflicted with osteoporosis or osteopenia. WNT signaling has emerged as a promising target pathway for developing novel bone anabolic therapeutics. Identifying bone-promoting WNT ligands and elucidating the underlying mechanisms may lead to useful therapeutic targets. The present study reports that WNT7B potently enhances bone formation through activation of mTORC1 in the mouse.

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of (64)Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. (64)Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated (64)Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, (64)Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.

Anogenital malignancy has a significant association with high-risk mucosal alpha-human papillomaviruses (alpha-PV), particularly HPV 16 and 18 whereas extragenital SCC has been linked to the presence of cutaneous beta and gamma-HPV types. Vulval skin may be colonised by both mucosal and cutaneous (beta-, mu-, nu- and gamma-) PV types, but there are few systematic studies investigating their presence and their relative contributions to vulval malignancy. Dysregulation of AKT, a serine/threonine kinase, plays a significant role in several cancers. Mucosal HPV types can increase AKT phosphorylation and activity whereas cutaneous HPV types down-regulate AKT1 expression, probably to weaken the cornified envelope to promote viral release. We assessed the presence of mucosal and cutaneous HPV in vulval malignancy and its relationship to AKT1 expression in order to establish the corresponding HPV and AKT1 profile of normal vulval skin, vulval intraepithelial neoplasia (VIN) and vulval squamous cell carcinoma (vSCC). We show that HPV16 is the principle HPV type present in VIN, there were few detectable beta types present and AKT1 loss was not associated with the presence of these cutaneous HPV. We show that HPV16 early gene expression reduced AKT1 expression in transgenic mouse epidermis. AKT1 loss in our VIN cohort correlated with presence of high copy number, episomal HPV16. Maintained AKT1 expression correlated with low copy number, an increased frequency of integration and increased HPV16E7 expression, a finding we replicated in another untyped cohort of vSCC. Since expression of E7 reflects tumour progression, these findings suggest that AKT1 loss associated with episomal HPV16 may have positive prognostic implications in vulval malignancy.

Small GTPase Ras homologue enriched in brain (RHEB) binds and activates the key metabolic regulator mTORC1, which has an important role in cancer cells, but the role of RHEB in cancer pathogenesis has not been shown. By performing a meta-analysis of published cancer cytogenetic and transcriptome databases, we defined a gain of chromosome 7q36.1-q36.3 containing the RHEB locus, an overexpression of RHEB mRNA in several different carcinoma histotypes, and an association between RHEB upregulation and poor prognosis in breast and head and neck cancers. To model gain of function in epithelial malignancy, we targeted Rheb expression to murine basal keratinocytes of transgenic mice at levels similar to those that occur in human squamous cancer cell lines. Juvenile transgenic epidermis displayed constitutive mTORC1 pathway activation, elevated cyclin D1 protein, and diffuse skin hyperplasia. Skin tumors subsequently developed with concomitant stromal angio-inflammatory foci, evidencing induction of an epidermal hypoxia-inducible factor-1 transcriptional program, and paracrine feed-forward activation of the interleukin-6–signal transducer and activator of transcription 3 pathway. Rheb-induced tumor persistence and neoplastic molecular alterations were mTORC1 dependent. Rheb markedly sensitized transgenic epidermis to squamous carcinoma induction following a single dose of Ras-activating carcinogen 7,12-dimethylbenz(a)anthracene. Our findings offer direct evidence that RHEB facilitates multistage carcinogenesis through induction of multiple oncogenic mechanisms, perhaps contributing to the poor prognosis of patients with cancers overexpressing RHEB. Cancer Res; 70(8); 3287–98. ©2010 AACR.

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

The construction of transgenic FVB/N mice targeting the PMLRARA fusion gene under the control of a human MRP8 promoter recapitulated the phenotype of acute promyelocytic leukemia but had the unexpected result of multiple squamous papillomas of the skin (Brown et al., PROC: Natl. Acad. Sci. USA, 94:2551-2556, 1997). In addition, transgenic MRP8-PMLRARA mice exhibited a skin phenotype characteristic of vitamin A deficiency. The severity of the skin phenotype and spontaneous papilloma development correlated with the level of transgene expression. Papilloma formation was preceded by follicular hyperplasia and the expression of epidermal differentiation markers in the follicular epithelium. Mutations in the Ha or Ki alleles of ras were not detected in papillomas that developed on transgenic skin, and papilloma formation was accentuated on the C57/Bl6 background, unlike the usual resistance of this strain to skin tumor induction. Analysis of liver extracts from transgenic mice indicated a deficiency in the production of retinoic acid. Furthermore, affected transgenic epidermis had reduced levels of retinoic acid receptoralpha (RARalpha) and retinoic X receptor (RXRalpha), and supplementation with exogenous retinoic acid prevented the skin phenotype. When transgenic keratinocytes were grafted to nude mice, the resulting integument was normal, and conversely, when transgenic bone marrow was grafted to normal mice, a skin phenotype did not develop. Together these results suggest that local interruption of PML and RARalpha signaling in the skin, together with a systemic retinoid deficiency, initiates a tumor induction pathway that is independent of ras activation.

The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human epidermis and in hyperproliferation conditions, including cancers, in adult epidermis. Published 2002 Wiley-Liss, Inc.

The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins1. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote2,3,4 and inhibit4,5,6 cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components, Skp1, Cul-1 (Cdc53 in yeast) and Rbx1, and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation2,3,5,7,8,9,10,11,12,13. To study the role of Cul-1 in mammalian development and cell-cycle regulation, we generated mice deficient for Cul1 and analysed null embryos and heterozygous cell lines. We show that Cul1 is required for early mouse development and that Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis.

New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

Resting energy expenditure (REE), nutritional parameters, and substrate levels were measured using a technique to minimize artifacts in 11 control patients, nine patients with localized, and four patients with diffuse neoplastic disease. Patients with diffuse disease had significantly increased percentage preillness weight loss (15.4 +/- 7.0%), decreased arm muscle circumference (21.9 +/- 2.1 vs. 26.2 +/- 3.5 cm), serum albumin (3.6 +/- 0.6 vs. 4.4 +/- 0.2 g/dl), total lymphocyte count (1024 +/- 613 vs. 1796 +/- 495 cells/mm3), and creatinine-height index (0.68 +/- 0.16 vs. 1.18 +/- 0.37) compared to controls. Both groups of cancer patients had significantly increased REE compared to controls: 25.6 +/- 3.7 (diffuse), 21.4 +/- 3.7 (localized), vs. 18.1 +/- 2.9 kcal/kg/d (controls). However, when REE was expressed as a function of metabolic body size, the significant difference persisted only in the patients with diffuse disease compared to controls: 71.8 +/- 16.4 vs. 53.9 +/- 8.1 kcal/kg3/4/d. Patients studied pre- and posttumor resection all had a postoperative drop in their REE, which was significantly correlated with the measured tumor volume. In this homogeneous, select group of patients, the tumor-bearing state exerts a moderate impact on nutritional and metabolic parameters, which are probably related to the extent of disease.

Deep venous thromboembolism (DVT) was studied in 44 patients with clinical Stage I, II, and III melanoma undergoing staging and therapeutic inguinal lymph node dissection. The ability of two noninvasive methods of surveillance, the phleborheograph (PRG) and the 125I fibrinogen scan to detect deep venous thrombosis was determined by comparison with prospective bilateral lower extremity venograms. In addition, the therapeutic impact, both beneficial and detrimental, of low dose heparin, 5000 units administered subcutaneously two hours prior to and every eight hours after operation was determined in a double blind study. The sensitivity of the PRG and 125I fibrinogen scan were both 20%. There were five deep venous thrombi, and two pulmonary emboli for a combined incidence of DVT of 13.6% for the entire patient population. However, there was no significant difference in the incidence of DVT between the two groups. The heparin-treated patients had an increased total volume (796 +/- 516 versus 388 +/- 208 ml; p less than 0.05), and duration of wound drainage (9 +/- 4 versus 13 +/- 6 days; p less than 0.05).

The incidence and severity of wound complications were examined in 105 patients with extremity and superficial truncal sarcomas who were eligible for wide local excision with or without adjuvant perioperative brachytherapy (BRT) and/or chemotherapy. Fifty-four cases from the eligible group were entered into a randomized prospective trial of the efficacy of BRT in decreasing local recurrence. In the eligible patients, major wound complications occurred in nine of 41 (22%) of the BRT cases, compared with two of 64 (3%) of the non-BRT patients, which was a significant increase (P = .002). The combined frequency of major and moderate wound complications was also significantly increased in the BRT (18 of 41, 44%) compared with the non-BRT (nine of 64, 14%) patients (P = .0006). The median duration to complete resolution of these complications was 189 days (14 to 597) in the BRT, compared with 49 (11 to 170) days in the non-BRT group (P = .0005); however, no amputations were required, and only 14% of the BRT-associated wound complications were of prolonged duration, ie, greater than 200 days. In the randomized study, both the total number of complications, and the combination of major and moderate complications were increased significantly in the BRT v the non-BRT patients. Adjuvant Adriamycin (Adria Laboratories, Columbus, OH) administered in 60 mg/m2 increments to a cumulative dose of 540 mg/m2 did not appear to impair wound healing even when administered within 15 days of operation. Significant wound complications occur in major resections of extremity and superficial truncal sarcomas. If the addition of adjuvant BRT produces a decrease in local recurrence, then either patient selection will have to be more rigidly applied, especially in wounds where skin flap blood supply is tenuous, or the technique will need to be modified to balance the short-term aim of reducing wound complications with the long-term goal of local tumor control.