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Non-small cell lung cancer (NSCLC) is the most frequent subtype of lung cancer and remains a highly lethal malignancy and one of the leading causes of cancer-related deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histologic form of NSCLC. In this study, we examined the role of PKCϵ, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Database analysis revealed an association between PKCϵ expression and poor outcome in patients with lung adenocarcinoma specifically harboring KRAS mutations. A PKCϵ-deficient, conditionally activatable allele of oncogenic Kras (LSL-KrasG12D;PKCϵ-/- mice) demonstrated the requirement of PKCϵ for Kras-driven lung tumorigenesis in vivo, which was consistent with impaired transformed growth reported in PKCϵ-deficient KRAS-dependent NSCLC cells. Moreover, PKCϵ-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations in Kras. Mechanistic analysis using RNA sequencing revealed little overlap for PKCϵ and KRAS in the control of genes and biological pathways relevant in NSCLC, suggesting that a permissive role of PKCϵ in KRAS-driven lung tumorigenesis may involve nonredundant mechanisms. Our results thus, highlight the relevance and potential of targeting PKCϵ for lung cancer therapeutics. Significance: These findings demonstrate that KRAS-mediated tumorigenesis requires PKCϵ expression and highlight the potential for developing PKCϵ-targeted therapies for oncogenic RAS-driven malignancies., Facultad de Ciencias Médicas, Centro de Investigaciones Inmunológicas Básicas y Aplicadas

Non-small cell lung cancer (NSCLC), the most frequent subtype of lung cancer, remains a highly lethal malignancy and one of the leading causes of cancer deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histological form of NSCLC. In this study, we examined the role of PKCepsilon, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Notably, database analysis revealed an association between PKCepsilon expression and poor outcome in lung adenocarcinoma patients specifically having KRAS mutation. By generating a PKCepsilon-deficient, conditionally activatable allele of oncogenic Kras (LSL-KrasG12D;PKCepsilon-/- mice) we were able to demonstrate the requirement of PKCepsilon for Kras-driven lung tumorigenesis in vivo, which is consistent with the impaired transformed growth observed in PKCepsilon-deficient KRAS-dependent NSCLC cells. Moreover, PKCepsilon-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations in Kras. Mechanistic analysis using RNA-Seq revealed little overlapping for PKCepsilon and KRAS in the control of genes/biological pathways relevant in NSCLC, suggesting that a permissive role of PKCepsilon in KRAS-driven lung tumorigenesis may involve non-redundant mechanisms. Our results thus highlight the relevance and potential of targeting PKCepsilon for lung cancer therapeutics., Centro de Investigaciones Inmunológicas Básicas y Aplicadas

The pro-oncogenic kinase PKCε is overexpressed in human prostate cancer and cooperates with loss of the tumor suppressor Pten for the development of prostatic adenocarcinoma. However, the effectors driving PKCε-mediated phenotypes remain poorly defined. Here, using cellular and mouse models, we showed that PKCε overexpression acts synergistically with Pten loss to promote NF-κB activation and induce cyclooxygenase-2 (COX-2) expression, phenotypic traits which are also observed in human prostate tumors. Targeted disruption of PKCε from prostate cancer cells impaired COX-2 induction and PGE2 production. Notably, COX-2 inhibitors selectively killed prostate epithelial cells overexpressing PKCε, and this ability was greatly enhanced by Pten loss. Long-term COX-2 inhibition markedly reduced adenocarcinoma formation, as well as angiogenesis in a mouse model of prostate-specific PKCε expression and Pten loss. Overall, our results provide strong evidence for the involvement of the canonical NF-κB pathway and its target gene COX2 as PKCε effectors, and highlight the potential of PKCε as a useful biomarker for the use of COX inhibition for chemopreventive and/or chemotherapeutic purposes in prostate cancer.

Colorectal cancer accounts for a substantial number of deaths each year worldwide. Lynch Syndrome is a genetic form of colorectal cancer (CRC) caused by inherited mutations in DNA mismatch repair (MMR) genes. Although researchers have developed mouse models of Lynch Syndrome through targeted mutagenesis of MMR genes, the tumors that result differ in important ways from those in Lynch Syndrome patients. We identified 60 cases of CRC in rhesus macaques (Macaca mulatta) at our facility since 2001. The tumors occur at the ileocecal junction, cecum and proximal colon and display clinicopathologic features similar to human Lynch Syndrome. We conducted immunohistochemical analysis of CRC tumors from several rhesus macaques, finding they frequently lack expression of MLH1 and PMS2 proteins, both critical MMR proteins involved in Lynch Syndrome. We also found that most macaque cases we tested exhibit microsatellite instability, a defining feature of Lynch Syndrome. Whole genome sequencing of rhesus macaque CRC cases identified mutations in MLH1 and/or MSH6 that are predicted to disrupt protein function. We conclude that this population of rhesus macaques constitutes a spontaneous model of Lynch Syndrome, matching the human disease in several significant characteristics, including genetic risk factors that parallel human Lynch Syndrome.

Significance The discovery of MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques (MLH1-rheMac HNPCC), which is an orthologue of Lynch syndrome in humans, is highly significant in the field of oncology. The hereditary nature of this disease should allow for planned cross-breeding of rhesus macaques to assess the effects of homozygous versus heterozygous MLH1 gene mutations, as well as other comutations and environmental factors that may affect the development of colon cancers. Also, the MLH1-rheMac HNPCC syndrome in rhesus macaques can serve as an important model for development of novel approaches to diagnosis and therapy of Lynch syndrome in human patients., Over the past two decades, 33 cases of colonic adenocarcinomas have been diagnosed in rhesus macaques (Macaca mulatta) at the nonhuman primate colony of the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center. The distinctive feature in these cases, based on PET/computed tomography (CT) imaging, was the presence of two or three tumor lesions in different locations, including proximal to the ileocecal juncture, proximal to the hepatic flexure, and/or in the sigmoid colon. These colon carcinoma lesions selectively accumulated [18F]fluorodeoxyglucose ([18F]FDG) and [18F]fluoroacetate ([18F]FACE) at high levels, reflecting elevated carbohydrate and fatty acid metabolism in these tumors. In contrast, the accumulation of [18F]fluorothymidine ([18F]FLT) was less significant, reflecting slow proliferative activity in these tumors. The diagnoses of colon carcinomas were confirmed by endoscopy. The expression of MLH1, MSH2, and MSH6 proteins and the degree of microsatellite instability (MSI) was assessed in colon carcinomas. The loss of MLH1 protein expression was observed in all tumors and was associated with a deletion mutation in the MLH1 promoter region and/or multiple single-nucleotide polymorphism (SNP) mutations in the MLH1 gene. All tumors exhibited various degrees of MSI. The pedigree analysis of this rhesus macaque population revealed several clusters of affected animals related to each other over several generations, suggesting an autosomal dominant transmission of susceptibility for colon cancer. The newly discovered hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques, termed MLH1-rheMac, may serve as a model for development of novel approaches to diagnosis and therapy of Lynch syndrome in humans.

C1q nephropathy was first described in 1985 as a process of glomerulonephritis with mesangial C1q deposit. The histology is similar to lupus nephritis, and was initially described as being seronegative renal lupus. However, these two entities are now considered different pathological processes. Its association with rheumatoid arthritis is unusual, and there are no cases with a similar presentation reported in the literature. In this article, the case is presented of a man who developed both these conditions.

Introducción. La hiponatremia es la alteración electrolítica más frecuente en pacientes con falla cardíaca crónica, que afecta hasta el 28% de pacientes en el ingreso hospilatario. Se ha demostrado que es un predictor independiente de mortalidad, estancia prolongada y rehospitalización. El objetivo es describir la frecuencia de hiponatremia en pacientes con falla cardíaca descompensada y su relación con la estancia hospitalaria y mortalidad. Metodología. Cohorte prospectiva en pacientes hospitalizados por el servicio de Medicina Interna en un hospital de cuarto nivel de Bogotá, Colombia, con diagnóstico de falla cardíaca descompensada entre abril de 2011 y marzo de 2012. Se midió el sodio al ingreso, a las 72 horas y al día séptimo de hospitalización. Se evaluó la relación de la hiponatremia al ingreso con la estancia hospitalaria y la mortalidad intrahospitalaria a 30 días. Resultados. Se incluyeron 162 pacientes, con promedio de edad de 72 años, 52.5% hombres. Cuarenta y uno (25.3%) presentaron hiponatremia al ingreso. Entre los pacientes que presentaron sodio normal al ingreso, 6 presentaron hiponatremia a las 72 horas y 2 a los 7 días. La mortalidad intrahospitalaria en la población total fue 9.2%, en el grupo de hiponatremia 12.2%, y 8.2% en los que no la tuvieron (p= 0.45). El promedio de estancia hospitalaria fue superior en los pacientes con hiponatremia, 6.6 vs. 8.4 días (p= 0.12). Discusión. La incidencia de hiponatremia al ingreso en este grupo de pacientes fue semejante a la reportada en la literatura. Conclusiones. Existe una tendencia hacia una mayor mortalidad y estancia hospitalaria en pacientes con hiponatremia, aun cuando no fue estadísticamente significativa. Introduction. Hyponatremia is the most common electrolyte disorder in patients with chronic heart failure affecting up to 28% of patients at admission. It has been shown to be an independent predictor of mortality, extended length of hospital stay and rehospitalization. The objective is to evaluate the presence of hyponatremia in patients with decompensated heart failure and describe its relationship with length of hospital stay and mortality. Methodology. A prospective cohort study in patients hospitalized at internal medicine service at a high level of complexity hospital in Bogotá, Colombia diagnosed with decompensated heart failure between April 2011 and March 2012. Sodium was measured on admission, 72 hours and at the seventh day of hospitalization. We evaluated the association between hyponatremia at hospital admission, mortality at 30 days and length of hospital stay. Results. 162 patients were included with an average age of 72 years, 52.5% male. Forty-one (25.3%) had hyponatremia at admission. Among the patients with normal sodium at admission, 6 had hyponatremia at 72 hours and 2 at the seventh day. In-hospital mortality in the total population was 9.2%, in the hyponatremia group 12.2%, and 8.2% in those who did not have it (p = 0.45). The average length of hospital stay was higher in patients with hyponatremia, 6.6 vs 8.4 days (p = 0.12). Discussion. The incidence of hyponatremia at admission in this group of patients was similar to that reported in the literature. Conclusions. The results showed a trend towards higher mortality and hospital stay in patients with hyponatremia, even though it was not statistically significant. Introdução. A hiponatremia é o desequilíbrio hidroeletrolítico mais frequente em pacientes com insuficiência cardíaca crônica, que afeta até 28% dos pacientes internados. Demonstrou ser um preditor independente de mortalidade, permanência prolongada e re-internação. O objetivo é descrever a frequência de hiponatremia em pacientes com insuficiência cardíaca descompensada e sua relação com internação hospitalar e mortalidade. Metodologia.Coorte prospectiva em pacientes internados pelo serviço de Medicina Interna em um hospital de quarto nível em Bogotá, Colômbia, com diagnóstico de insuficiência cardíaca descompensada, entre abril de 2011 e março de 2012. Foi medido o sódio na admissão, às 72 horas e no sétimo dia de internação. Foi avaliada a relação de hiponatremia na admissão com internação e a mortalidade hospitalar aos 30 dias. Resultados. Foram incluídos 162 pacientes, com idade média de 72 anos, 52,5% homens. Quarenta e um (25,3%) apresentaram hiponatremia na admissão. Entre os pacientes que apresentaram sódio normal na admissão, seis apresentaram hiponatremia às 72 horas, e dois aos sete dias. A mortalidade hospitalar na população total foi de 9,2%, no grupo de hiponatremia 12,2% e 8,2% naqueles que não apresentaram (p = 0,45). O tempo médio de internação foi maior nos pacientes com hiponatremia, 6,6 vs. 8,4 dias (p = 0,12). Discussão. A incidência de hiponatremia na admissão nesse grupo de pacientes foi semelhante à relatada na literatura. Conclusões. Existe uma tendência de maior mortalidade e permanência hospitalar em pacientes com hiponatremia, mesmo que não tenha sido estatisticamente significante.

The guinea pig, as a livestock species, is still developing and growing throughout Peru and neighboring countries, as reflected by its increasing export since 2000. However, the selection of proven fertile males is tedious due to the absence of seminal parameter standards and the lack of safe semen collection techniques. Thus, pregnancy detection or live births are required for males&rsquo, selection. The purpose of this study was to describe the qualitative and quantitative semen parameters of fertile guinea pig males, to set reference values, and to validate a novel electroejaculation technique for the species. Semen was collected at weekly intervals from sixteen fertile males. Four transrectal electroejaculations were performed per male with 95% successful collections, yielding 39 viable semen samples. Seminal characteristics were as follows: pH 7.0 &plusmn, 0.13, ejaculate volume 0.67 &plusmn, 0.55 mL, sperm motility 90.81 &plusmn, 6.64%, sperm concentration 36.7 &plusmn, 28.41 &times, 106 sperm/mL, sperm count 20.09 &plusmn, 17.56 &times, 106 sperm/ejaculate, percentage of abnormal morphology 18.26 &plusmn, 8.52%, and percentage ubiquitinated spermatozoa 5.57 &plusmn, 6.28%. These values will serve as a reference to detect best breeding and infertile males rapidly. The described techniques are reproducible by commercial producers.

International audience; Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.

PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB) pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment., Centro de Investigaciones Inmunológicas Básicas y Aplicadas

Protein kinase C ϵ (PKCϵ) has emerged as an oncogenic kinase and plays important roles in cell survival, mitogenesis and invasion. PKCϵ is up-regulated in most epithelial cancers, including prostate, breast, and lung cancer. Here we report that PKCϵ is an essential mediator of NF-κB activation in prostate cancer cells. A strong correlation exists between PKCϵ overexpression and NF-κB activation status in prostate cancer cells. Moreover, transgenic overexpression of PKCϵ in the mouse prostate causes preneoplastic lesions that display significant NF-κB hyperactivation. PKCϵ RNAi depletion or inhibition in prostate cancer cells diminishes NF-κB translocation to the nucleus with subsequent impairment of both activation of NF-κB transcription and induction of NF-κB responsive genes in response to the proinflammatory cytokine tumor necrosis factor α (TNFα). On the other hand, PKCϵ overexpression in normal prostate cells enhances activation of the NF-κB pathway. A mechanistic analysis revealed that TNFα activates PKCϵ via a C1 domain/diacylglycerol-dependent mechanism that involves phosphatidylcholine-phospholipase C. Moreover, PKCϵ facilitates the assembly of the TNF receptor-I signaling complex to trigger NF-κB activation. Our studies identified a molecular link between PKCϵ and NF-κB that controls key responses implicated in prostate cancer progression.

Lysosomal cysteine protease cathepsin L (CTSL) is believed to play a role in tumor progression and is considered a marker for clinically invasive tumors. Studies from our laboratory using the classical mouse skin carcinogenesis model, with 7,12-dimethyl-benz[a]anthracene (DMBA) for initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) for promotion, showed that expression of CTSL is increased in papillomas and squamous cell carcinomas (SCC). We also carried out carcinogenesis studies using Ctsl-deficient nackt (nkt) mutant mice on three different inbred backgrounds. Unexpectedly, the multiplicity of papillomas was significantly higher in Ctsl-deficient than in wild-type mice on two unrelated backgrounds. Topical applications of TPA or DMBA alone to the skin of nkt/nkt mice did not induce papillomas, and there was no increase in spontaneous tumors in nkt/nkt mice on any of the three inbred backgrounds. Reduced epidermal cell proliferation in Ctsl-deficient nkt/nkt mice after TPA treatment suggested that they are not more sensitive than wild-type mice to TPA promotion. We also showed that deficiency of CTSL delays terminal differentiation of keratinocytes, and we propose that decreased elimination of initiated cells is at least partially responsible for the increased papilloma formation in the nackt model.

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCε, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCα, PKCδ, or PKCε in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCε and PB-PKCδ mice did not show any evident phenotype, PB-PKCε mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCε overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCε by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCε overexpression and prostate cancer development.

International audience; Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.

In the past 20 yr, several inbred strains have been derived from SENCAR outbred mice. These strains display different susceptibility to the induction of papillomas and progression to squamous cell carcinomas (SCC) in the skin after chemical carcinogenesis. In the present study, we showed that one of these strains SENCARB/Pt was highly susceptible to the development of N-methyl-N-nitrosourea (MNU)- and 7,12-dimethylbenz[a]anthracene (DMBA)-induced lymphomas. In contrast, the SSIN/Sprd inbred strain is completely resistant to T-cell lymphomagenesis by both carcinogens. Within 175 d after a single injection of 75 mg/kilogram body weight (kbw) of MNU, SENCARB/Pt mice exhibited a 91.6% incidence of lymphoma. In addition, during an independent tumorigenesis study with repeated doses of intragastric DMBA, SENCARB/Pt mice showed an incidence of 75% lymphoma development 300 d after the last treatment. Histopathological and flow cytometric parameters indicated that the lymphomas were of the T-cell lineage. In order to study the genetics of MNU-induced tumorigenesis, we generated F1 hybrid mice between SSIN/Sprd and SENCARB/Pt mice. Tumor incidence in MNU-injected F1 mice suggested that the high tumor incidence is a dominant trait. Loss of heterozygosity (LOH) analysis in these tumor samples revealed allelic imbalances on chromosomes 15 and 19. Given that these inbred strains are closely related, it is likely that a relatively small number of loci are responsible for the observed differences in susceptibility. Therefore, these SENCAR inbred strains constitute important new tools to study the genetic basis of resistance and susceptibility to chemically induced thymic lymphoma formation.

Squamous carcinomas of the head and neck (HNSCC) represent the sixth most common cancer among men worldwide and a major cause of morbidity and mortality due to its relatively poor prognosis. As part of ongoing studies addressing the molecular events underlying tumor progression in HNSCC, we have explored the nature of the proliferative pathways in which dysregulation may promote aberrant cell growth in this tumor type. The serine/threonine protein kinase Akt is a downstream target of phosphatidylinositol 3-kinase and a key regulator of normal and cancerous growth and cell fate decisions. Therefore, in this study, we have examined the status of activation of Akt in different stages of squamous cell carcinoma development in mice and in clinical samples from HNSCC patients. By immunohistochemical analysis, using a recently developed phosphorylation state-specific antibody, we demonstrated that Akt activation correlates closely with the progression of mouse skin squamous cell carcinoma. We also observed that activation of Akt is a frequent event in human HNSCC because active Akt can be detected in these tumors with a pattern of expression and localization correlating with the progression of the lesions. In line with these observations, Akt was constitutively activated in a large fraction of HNSCC-derived cell lines. We also provide evidence that the Akt signaling pathway may represent a biologically relevant target for a novel antineoplastic agent, UCN-01, which recently has been shown to be active in cellular and xenograft models for HNSCC at concentrations safely achievable in clinically relevant situations.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.

Protein Kinase C epsilon (PKCϵ), a pro-oncogenic member of the PKC family of phorbol ester/diacylglycerol receptors, has been shown to be up-regulated in most epithelial cancers, including prostate cancer. PKCϵ transgenic overexpression in the mouse prostate causes preneoplastic lesions, however when intercrossed with mice haploinsufficient for Pten, the resulting compound mice (PB-PKCϵ;Pten+/-) develops invasive adenocarcinoma. We found that Pten-deficient prostate cell lines engineered to overexpress PKCϵ (CaP8-PKCϵ) cells become highly invasive and tumorigenic. Microarray gene expression profiling in this cell line revealed marked changes in the expression of genes implicated in proliferation, migration, adhesion, EMT, angiogenesis and metabolism. In particular, the chemokine CXCL13 was identified as the top-most up-regulated gene in CaP8-PKCϵ cells. Measurements of either CXCL13 mRNA, protein release to the culture medium (CM), or Cxcl13 promoter activity showed clear enhancements in cells overexpressing PKCϵ or Pten depleted, and these effects were enriched synergistically in CaP8-PKCϵ cells. CaP8-PKCϵ subjected to CXCL13 RNAi depletion showed reduced motility and tumorigenic growth. Similar results were observed upon silencing of CXCR5, the CXCL13 receptor. CXCL13 expression and promoter activity in CaP8-PKCϵ cells were significantly reduced by GF109203X (PKC inhibitor), IKK16, wedelolactone (NF-κB inhibitors), and BKM120 (PI3K inhibitor), but were insensitive to BX795 (PDK1 inhibitor). RNAi-mediated depletion of NIK or IKKα significantly blunted Cxcl13 luciferase promoter activity in CaP8-PKCϵ cells, but the effect was partially inhibited by IKKβ RNAi, suggesting the role of the atypical NF-κB pathway. Mutation of a responsive element for atypical NF-κB in the CXCL13 promoter abolished CXCL13 reporter activity in CaP8-PKCϵ cells. Taken together, our study identifies a novel paradigm depicting atypical NF-κB as the transcriptional signaling pathway by which PKCϵ overexpression/Pten loss regulates overproduction of the chemokine CXCL13 in prostate cancer cells. Citation Format: Rachana Garg, Jorge Blando, Carlos Perez, Martin Abba, Fernando Benavides, Marcelo Kazanietz. Oncogenic PKC epsilon and Pten loss converge on activating CXCL13 via atypical NF-κB transcriptional signaling in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-230. doi:10.1158/1538-7445.AM2017-LB-230

From the standpoint of evolution, diploidy is generally considered advantageous for two reasons. First, because diploid organisms possess twice as many genes as haploids and in these conditions twice as many favorable mutations arise per generation. This of course increases the genetic diversity in the population and, finally, contributes to the progress of adaptive evolution. Diploidy is also considered advantageous because, when a recessive mutation occurs in a given gene, there is always a backup copy of the original allele on the other chromosome, offering a chance for the population to assess, with no risk, which one of the two alleles is most advantageous for the future of the species in a given environmental context. In most cases, the new mutant allele is neutral and has no selective advantage; sometimes it is harmful and is more or less rapidly eliminated. On rare occasions, it is beneficial and can then gradually replace the original allele.

Four adult mutant nackt mice, which demonstrate alopecia and CD4+ T-cell deficiency, and two outbred SENCAR mice (sentinels) were presented for routine health surveillance. Lesions were not observed at necropsy. Microscopically, all four nackt mice demonstrated multiple concretions that were 30–100 μm in diameter, irregularly rounded to oval, nonbirefringent, and amphophilic to basophilic. Many of the concretions appeared attached to or within the alveolar walls of all lung lobes. Approximately half of the concretions had irregular fracture lines. All concretions were periodic acid-Schiff positive, and Von Kassa staining revealed diffuse calcification. None of the concretions were associated with inflammatory cell infiltrates, and metaplastic ossification was not evident. A diagnosis of pulmonary alveolar microlithiasis, a rare disease in both humans and animals, was made based on the size and location of the concretions and the lack of an inflammatory response. This is the first report of a laboratory mouse demonstrating pulmonary alveolar microlithiasis.

Twenty-five colon carcinoma cases from a specific pathogen free (SPF) breeding colony of Indian origin rhesus macaques (Macaca mulatta) were examined and evaluated for pathogenesis. Although, no sp...

The tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten(+/-) heterozygotes in either lesion development or progression. Lesions in both Pten(+/-); Tsc2(+/-), and Pten(+/-) mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten(+/-) mice. Furthermore, prostate lesion growth in Pten(+/-) mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment.

Prostate cancer is one of the most commonly diagnosed malignancies and the second leading cause of cancer-related deaths among men in the United States. Protein kinase C epsilon (PKCϵ), a member of the PKC family of phorbol ester/diacylglycerol receptors, has emerged as an oncogenic kinase and shown to be up-regulated in human prostate cancer specimens. We recently demonstrated that PKCϵ is an upstream regulator of NF-κB activation in prostate cancer (JBC, 287:37570-37582, 2012) and that transgenic overexpression of PKCϵ in mice prostate under the control of the probasin promoter (PB-PKCϵ) leads to hyperplasia and PIN lesions but was insufficient to drive neoplastic changes (Cell Cycle, 10:268-277, 2011). Notably, when we intercrossed PB-PKCϵ mice with mice haploinsufficient for Pten, another common genetic alteration in human prostate cancer, the resulting compound mutant mice (PB-PKCϵ;Pten+/- mice) developed fully invasive adenocarcinoma with elevated NF-κB levels. In the present study, we aim to delineate the mechanism underlying the observed cooperativity between PKCϵ overexpression and Pten deficiency and to explore the consequences of this cooperativity on the transcription factor NF-κB signaling, a pathway known to be highly dysregulated in prostate tumorigenesis. To this end, we stably overexpressed PKCϵ in mouse prostate epithelial lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion. We observed a striking synergism between PKCϵϵ overexpression and Pten loss in conferring enhanced proliferative, migratory and invasive phenotype. Moreover, LPS or TNFα stimulation of these cells led to increased NF-κB activation as evident from the elevated IκBα phosphorylation, NF-κB nuclear translocation and transactivation of a NF-κB luciferase reporter. These effects were much more pronounced in CaP8 cells. Of note, PKCϵ overexpression and Pten loss also cooperates to augment levels of the NF-κB regulated gene, COX-2. Stable overexpression of PKCϵ and Pten depletion in “normal” immortalized RWPE1 cells also resulted in significant enhancements in TNFα-induced NF-κB activation and COX-2 induction. Furthermore, NF-κB inhibition by parthenolide significantly retarded the growth of CaP8-PKCϵ tumors in athymic nude mice. Overall, our results identify NF-κB as a mediator of PKCϵ oncogenesis in prostate cancer, particularly in the context of Pten loss. Citation Format: Rachana Garg, Jorge Blando, Carlos J. Perez, Fernando J. Benavides, Marcelo G. Kazanietz. Protein kinase C ϵ cooperates with Pten deficiency to regulate NF-κB pathway in prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2015-799

The levels of 8S-lipoxygenase (8S-LOX) expression and of its arachidonic acid metabolite, 8-hydroxyeicosatetraenoic acid (8-HETE), are highly elevated in the early stages of mouse skin carcinogenesis. On the other hand, several reports showing that 8-HETE is also closely associated with keratinocyte differentiation raise a question concerning the role of 8S-LOX/8-HETE in skin carcinogenesis. To address that question, here we conducted a series of gain-of-function studies. Skin targeted loricrin 8S-LOX/C57BL/6J transgenic mice showed a more differentiated epidermal phenotype as well as a 64% reduced papilloma development in a two-stage skin carcinogenesis protocol. Forced expression of 8S-LOX in MT1/2 cells, a murine papilloma cell line, also caused a more differentiated appearance as well as keratin 1 expression. Overexpression of 8S-LOX in CH72 cells, a murine carcinoma cell line, inhibited cell proliferation by 30% in vitro and by 86% in in vivo xenografts. Exogenous addition of 5 μ M 8-HETE to CH72 cells caused cell cycle arrest at the G1 phase. Finally, immunohistochemical analyses showed 8S-LOX protein expression was strictly confined to the differentiated compartment of mouse skin and throughout tumorigenesis. Collectively, these data suggest that 8S-LOX plays a role as a prodifferentiating, antitumorigenic, and tumor suppressing gene in mouse skin carcinogenesis.

Susceptibility to tumor development varies among individuals in the human population. This variability can also be found among different strains of mice, particularly in the mouse skin chemical carcinogenesis model. The genetic mechanisms underlying mouse skin tumor susceptibility are not fully understood. The SENCAR stock has been found to be the most sensitive mice for skin carcinogenesis studies; however, little is known about the genes underlying tumor susceptibility, particularly, those involved in tumor progression. Experiments with the SSIN/Sprd mice, an inbred strain derived from the outbred SENCAR stock, suggested that papilloma development, tumor promotion, and their conversion into squamous cell carcinomas (SCCs), progression, are regulated by different genes. In the highly sensitive SSIN/Sprd mice, papillomas rarely progress to SCC. Using crosses between the outbred SENCAR and the SSIN/Sprd mice, we previously determined that papilloma progression in the SENCAR stock could be controlled by at least one autosomal dominant gene. However, the outbred nature of the SENCAR stock precluded us from extending those findings. More recently, another inbred strain was developed from the outbred SENCAR stock, the SENCARB/Pt. These mice have similar tumor promotion sensitivity to the SSIN/Sprd but in contrast, have high papilloma progression susceptibility, similar to the outbred original stock. In the present study, we generated F(1), F(2), and backcross hybrids between the SSIN/Sprd and SENCARB/Pt mice to determine a possible model for tumor progression susceptibility and to map the putative tumor susceptibility genes. Our tumor data suggests that papilloma progression susceptibility in the SENCAR mouse skin model could be genetically determined by one susceptibility gene. Our preliminary linkage analysis failed to identify one strong susceptibility locus to confirm this but provided some evidence for at least one possible susceptibility locus in mouse chromosome 14.

A spontaneous recessive mutation named nackt (symbol: nkt) affecting hair growth and T-cell development was discovered in a moderately inbred stock of mice. Skin lesions were characterized by sparse rough coat, bare patches around the eyes and neck, and a scratching behavior throughout life. Fluorescence-activated cell sorter analysis indicated a deficiency in the CD4(+) 8(-) T-cell subset in the thymus and a marked decrease in CD4(+) T cells in peripheral lymphoid organs. Linkage analysis using a set of molecular markers and an F2 intersubspecific cross indicated that the mutation maps to the central region of mouse chromosome 13, in a region homologous to human chromosome 5q22-q35.

Protein kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, is up-regulated in many human cancers, including prostate cancer. We recently demonstrated that PKCε is an essential mediator of NF-κB activation in prostate cancer (Garg et al., JBC, 287 (44), 37570–37582, 2012). Cyclooxygenase-2 (COX-2), a well-known NF-κB regulated gene, has been reported to be up-regulated in a number of human cancers, including metastatic prostate cancer. As PKCε plays an important role in prostate cancer cell survival and cooperates with other oncogenic insults, herein we aim to determine if PKCε regulates COX-2 activation during prostate tumorigenesis. PKCε RNAi depletion diminishes TNFα- or LPS-induced COX-2 mRNA expression in LNCaP prostate cancer cells and constitutive COX-2 levels from PC3 cells. Conversely, PKCε overexpression by adenoviral means potentiates TNFα or LPS-induced COX-2 expression in LNCaP cells. Notably, transgenic overexpression of PKCε in the mouse prostate causes preneoplastic lesions with elevated COX-2 levels. Interestingly, when we intercrossed the prostate-specific PKCε transgenic mice with mice haploinsufficient for Pten, a common genetic alteration in human prostate cancer, the resulting compound mutant mice (PB-PKCε;Pten+/− mice) developed fully invasive adenocarcinoma with NF-κB hyperactivation and high COX-2 levels. Likewise, stable overexpression of PKCε in mouse prostate epithelial cell lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion, led to significant enhancements in cell proliferation, motility and invasiveness as well as in LPS-induced COX-2 mRNA expression compared to the respective control cells, and this effect is more pronounced in CaP8 cells. Studies using human prostate tumors revealed a co-existence of PKCε overexpression, NF-κB hyperactivation, and COX-2 up-regulation. Lastly, treatment of PKCε overexpressing P8 or CaP8 cells with the selective COX-2 inhibitor NS398 caused a pronounced growth inhibition. Overall, our study identified COX-2 as a PKCε-regulated gene. Our results suggest that COX-2 as a potential mediator of PKCε oncogenesis in prostate cancer, particularly in the context of Pten loss. Citation Format: Rachana Garg, Priti Lal, Jorge Blando, Fernando J. Benavides, Michael D. Feldman, Emer M. Smyth, Marcelo G. Kazanietz. COX-2 as a mediator of oncogenic PKCε in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-154. doi:10.1158/1538-7445.AM2013-LB-154

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related deaths among men. Protein kinase C epsilon (PKC∈), a member of the PKC family of phorbol ester/diacylglycerol receptors, has been shown to be up-regulated in human prostate cancer specimens. We recently demonstrated that mice that specifically overexpress PKC∈ in prostate under the control of the probasin promoter (PB-PKC∈) develop hyperplasia and PIN lesions. However, PKC∈ overexpression was insufficient to drive neoplastic changes in the mouse prostate. In the present study our goal was to determine whether PKC∈ overexpression can cooperate with Pten deficiency, one of the most common genetic alterations in human prostate cancer. We stably overexpressed PKC∈ in two pairs of isogenic mouse prostate epithelial lines that are either heterozygous (P2, P8) or homozygous (CaP2, CaP8) for Pten deletion. PKC∈ overexpression led to significant enhancement in cell proliferation compared to the respective control cells and this effect is more pronounced in CaP2 or CaP8 cells. Moreover, assays of colony formation in liquid medium revealed that anchorage-dependent growth was augmented in PKC∈-overexpressing cells relative to control cells. A significant elevation in phospho-Akt levels was observed in PKC∈ overexpressing cells relative to respective controls. To further elucidate the cooperativity between PKC∈ overexpression and Pten loss we intercrossed the PB-PKC∈ transgenic mice and Pten+/− mice. Strikingly, the resulting compound mutant mice overexpressing PKC∈ and haploinsufficient for Pten (PB-PKC∈; Pten+/−) developed fully invasive adenocarcinoma at 12 months of age. Very strong phospho-Akt, phospho-S6 and phospho mTOR staining could be detected in adenocarcinomas from PB-PKC∈; Pten+/− mice. Furthermore, we detected enhanced p-Erk, Cox-2, STAT3, NF-κB and cyclin D1 signaling in adenocarcinomas from PB-PKC∈;Pten+/− mice than in lesions from each monogenic model. These data thus suggest that PKC∈ overexpression cooperates with a defined oncogenic input to promote prostate tumorigenesis. These findings also suggest a crucial role of PKC∈ in survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 534. doi:1538-7445.AM2012-534

Protein Kinase C epsilon (PKC∈), a member of the PKC family of phorbol ester/diacylglycerol receptors, has been shown to be up-regulated in human prostate cancer specimens. As PKC∈ plays an important role in prostate cancer cell survival and cooperates with other oncogenic insults, we hypothesized that PKC∈ may regulate NF-κB signaling pathway, known to be highly dysregulated during prostate tumorigenesis. Similar to human tumors, we observed high PKC∈ overexpression in prostate cancer cell lines compared to normal immortalized RWPE-1 cells. PKC∈ depletion using siRNA or its pharmacological inhibition in LNCaP cells resulted in decreased TNFα-induced IκBα phosphorylation and degradation, as well as decreased NF-κB nuclear translocation and transactivation of NF-κB reporter activity. Conversely, significant enhancements in these TNFα-induced responses were observed in LNCaP cells upon PKC∈ overexpression using an adenoviral approach. Interestingly, TNFα treatment led to activation of PKC∈ in LNCaP cells, as analyzed by enhanced translocation from cytosolic to membrane fractions. Furthermore, TNFα-mediated PKC∈ translocation and IκBα phosphorylation were found to be dependent on phosphatidylcholine- but not phosphatidylinositol-specific phospholipase C. As determined by co-immunoprecipitation, PKC∈ associates with TNF receptor-1 (TNFR1) in response to TNFα and has important regulatory consequences in the assembly of TNFR-1 signaling complex. PKC∈ depletion by siRNA decreased the TNFα- mediated recruitment of receptor interacting protein (RIP) and TNFR-associated factor-2 (TRAF2), which are both known to mediate NF-κB activation. In accordance with the co-immunoprecipitation results, siRNA-mediated PKC∈ depletion inhibits TNFR1, TRADD, TRAF2, IKK-β- mediated NF-κB reporter activity. Furthermore, effects of PKC∈ overexpression on NF-κB transcriptional activity correlated well with the increased mRNA levels of known NF-κB regulated genes involved in cell survival, inflammation, invasion and angiogenesis (Cox2, VEGF, MMP9, and IL8). Finally, we found that in preneoplastic lesions from transgenic mice overexpressing PKC∈ in the prostate there is enhanced NF-κB activation. In summary, our study established a molecular link between PKC∈ and NF-κB in prostate cancer and suggests a prominent role for this pathway in prostate cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2011-1031