Carlos J. Perez

Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCe, 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 PKCa, PKCd, or PKCe in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCa and PB-PKCd mice did not show any evident phenotype, PB-PKCe 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. PKCe overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCe 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 PKCe overexpression and prostate cancer development.

Activation of Nuclear Factor κB (NF-κB) in Prostate Cancer Is Mediated by Protein Kinase C ϵ (PKCϵ)

Background: PKCϵ, a potential oncogene, is up-regulated in prostate cancer. Results: PKCϵ facilitates the formation of TNFR-I complex to regulate the NF-κB pathway via a C1 domain/diacylglycerol-dependent mechanism. Conclusion: PKCϵ is an upstream regulator of NF-κB signaling in prostate cancer. Significance: Mechanisms identified here may reveal novel PKCϵ effectors that contribute to prostate cancer progression and highlight the potential relevance of this pathway for therapeutic purposes. 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.

Activation of Nuclear Factor-Kappa B (NFκB) in Prostate Cancer is Mediated by PKC Epsilon (PKCϵ)

Background: PKCϵ, a potential oncogene, is up-regulated in prostate cancer. Results: PKCϵ facilitates the formation of TNFR-I complex to regulate the NF-κB pathway via a C1 domain/diacylglycerol-dependent mechanism. Conclusion: PKCϵ is an upstream regulator of NF-κB signaling in prostate cancer. Significance: Mechanisms identified here may reveal novel PKCϵ effectors that contribute to prostate cancer progression and highlight the potential relevance of this pathway for therapeutic purposes. 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.

Two Hypomorphic Alleles of Mouse Ass1 as a New Animal Model of Citrullinemia Type I and Other Hyperammonemic Syndromes

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.

Increased susceptibility to skin carcinogenesis associated with a spontaneous mouse mutation in the palmitoyl transferase Zdhhc13 gene

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared to wild-type epidermis, in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than wild-type littermates. To our knowledge, this is the first report of a protective role for a PAT in skin carcinogenesis.

Several Classical Mouse Inbred Strains, Including DBA/2, NOD/Lt, FVB/N, and SJL/J, Carry a Putative Loss-of-Function Allele of Gpr84

G protein-coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains.

Leucine-rich repeat containing 8A (LRRC8A)–dependent volume-regulated anion channel activity is dispensable for T-cell development and function

Background: Leucine‐rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine‐rich repeats (LRRs) at its C‐terminal end and is an essential component of the volume‐regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T‐ and B‐cell development in mice. Lrrc8a−/− mice have severely defective T‐cell development and function. It is not known whether the T‐ and B‐cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. Objective: We sought to determine whether VRAC activity is required for normal T‐cell development and function. Methods: VRAC activity was examined by using patch‐clamp analysis. Flow cytometry was used to examine T‐cell development. T‐cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. Results: We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2‐bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a−/− mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a−/− mice, ebo/ebo mice have normal T‐cell development and function and intact antibody response to T‐dependent antigen. Conclusion: LRRC8A‐dependent VRAC activity is dispensable for T‐cell development and function.

Zdhhc13-dependent Drp1 S-palmitoylation impacts brain bioenergetics, anxiety, coordination and motor skills

Protein S-palmitoylation is a reversible post-translational modification mediated by palmitoyl acyltransferase enzymes, a group of Zn2+-finger DHHC-domain-containing proteins (ZDHHC). Here, for the first time, we show that Zdhhc13 plays a key role in anxiety-related behaviors and motor function, as well as brain bioenergetics, in a mouse model (luc) carrying a spontaneous Zdhhc13 recessive mutation. At 3 m of age, mutant mice displayed increased sensorimotor gating, anxiety, hypoactivity, and decreased motor coordination, compared to littermate controls. Loss of Zdhhc13 in cortex and cerebellum from 3- and 24 m old hetero- and homozygous male mutant mice resulted in lower levels of Drp1 S-palmitoylation accompanied by altered mitochondrial dynamics, increased glycolysis, glutaminolysis and lactic acidosis, and neurotransmitter imbalances. Employing in vivo and in vitro models, we identified that Zdhhc13-dependent Drp1 S-palmitoylation, which acting alone or in concert, enables the normal occurrence of the fission-fusion process. In vitro and in vivo direct Zdhhc13-Drp1 protein interaction was observed, confirming Drp1 as a substrate of Zdhhc13. Abnormal fission-fusion processes result in disrupted mitochondria morphology and distribution affecting not only mitochondrial ATP output but neurotransmission and integrity of synaptic structures in the brain, setting the basis for the behavioral abnormalities described in the Zdhhc13-deficient mice.

MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques.

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.

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

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.