Fernando Benavides

Persistent Activation of the Akt Pathway in Head and Neck Squamous Cell Carcinoma: A Potential Target for UCN-01

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.

The hairless mouse in skin research

The hairless (Hr) gene encodes a transcriptional co-repressor highly expressed in the mammalian skin. In the mouse, several null and hypomorphic Hr alleles have been identified resulting in hairlessness in homozygous animals, characterized by alopecia developing after a single cycle of relatively normal hair growth. Mutations in the human ortholog have also been associated with congenital alopecia. Although a variety of hairless strains have been developed, outbred SKH1 mice are the most widely used in dermatologic research. These unpigmented and immunocompetent mice allow for ready manipulation of the skin, application of topical agents, and exposure to UVR, as well as easy visualization of the cutaneous response. Wound healing, acute photobiologic responses, and skin carcinogenesis have been extensively studied in SKH1 mice and are well characterized. In addition, tumors induced in these mice resemble, both at the morphologic and molecular levels, UVR-induced skin malignancies in man. Two limitations of the SKH1 mouse in dermatologic research are the relatively uncharacterized genetic background and its outbred status, which precludes inter-individual transplantation studies.

Impaired Hair Follicle Morphogenesis and Cycling with Abnormal Epidermal Differentiation in nackt Mice, a Cathepsin L-Deficient Mutation

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.

PTEN deficiency is fully penetrant for prostate adenocarcinoma in C57BL/6 mice via mTOR-dependent growth.

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.

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.

Protective role of cathepsin L in mouse skin carcinogenesis.

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. Mol. Carcinog.

Nackt (nkt), a new hair loss mutation of the mouse with associated CD4 deficiency

Abstract 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.

An antitumorigenic role for murine 8S-lipoxygenase in skin carcinogenesis

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 μM 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.

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.