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Squamous cell carcinoma (SCC) of the skin is a keratinocyte malignancy characterized by tumors presenting on sun-exposed areas with surgery being the mainstay treatment. Despite advances in targeted therapy in other skin cancers, such as basal cell carcinoma and melanoma, there have been no such advances in the treatment of SCC. This is partly due to an incomplete knowledge of the pathogenesis of SCC. We have recently identified a protein kinase C-associated kinase (PKK) as a potential tumor suppressor in SCC. We now describe a novel conditional PKK knockout mouse model, which demonstrates that PKK deficiency promotes SCC formation during chemically induced tumorigenesis. Our results further support that PKK functions as a tumor suppressor in skin keratinocytes and is important in the pathogenesis of SCC of the skin. We further define the interactions of keratinocyte PKK with TP63 and NF-κB signaling, highlighting the importance of this protein as a tumor suppressor in SCC development.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies that can result in damage to multiple organs. It is well documented that B cells play a critical role in the development of the disease. We previously showed that protein kinase C associated kinase (PKK) is required for B1 cell development as well as for the survival of recirculating mature B cells and B-lymphoma cells. Here, we investigated the role of PKK in lupus development in a lupus mouse model. We demonstrate that the conditional deletion of PKK in B cells prevents lupus development in Sle1Sle3 mice. The loss of PKK in Sle mice resulted in the amelioration of multiple classical lupus-associated phenotypes and histologic features of lupus nephritis, including marked reduction in the levels of serum autoantibodies, proteinuria, spleen size, peritoneal B-1 cell population and the number of activated CD4 T cells. In addition, the abundance of autoreactive plasma cells normally seen in Sle lupus mice was also significantly decreased in the PKK-deficient Sle mice. Sle B cells deficient in PKK display defective proliferation responses to BCR and LPS stimulation. Consistently, B cell receptor-mediated NF-κB activation, which is required for the survival of activated B cells, was impaired in the PKK-deficient B cells. Taken together, our work uncovers a critical role of PKK in lupus development and suggests that targeting the PKK-mediated pathway may represent a promising therapeutic strategy for lupus treatment.

Protein kinase C associated kinase (PKK) regulates NF-κB activation and is required for the survival of certain lymphoma cells. Mice lacking PKK die soon after birth, and previous studies suggest that the role of PKK in B cell development might be context dependent. We have generated a mouse strain harboring conditional null alleles for PKK and a Cre-recombinase transgene under the control of the endogenous CD19 promoter. In the present study, we show that knockout of PKK in B cells results in the reduction of long-lived recirculating mature B cell population in lymph nodes and bone marrow as well as a decrease in peritoneal B1 cells, while PKK deficiency has no apparent effect on early B cell development in bone marrow or the development of follicular and marginal zone B cells in the spleen. In addition, we demonstrate that PKK-deficient B cells display defective proliferation and survival responses to stimulation of B cell receptor (BCR), which may underlie the reduction of recirculating mature B cells in PKK mutant mice. Consistently, BCR-mediated NF-κB activation, known to be required for the survival of activated but not resting B cells, is attenuated in PKK-deficient B cells. Thus, our results reveal a critical role of PKK in the maintenance of recirculating mature B cells as well as the development of B1 cells in mice.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

Non-melanoma skin cancer represents the most common cancer in the United States. Squamous cell carcinoma (SCC) of the skin is a subtype of NMSC that shows a greater potential for invasion and metastasis. The current study identifies the protein kinase C-associated kinase (PKK), which is also known as the receptor-interacting protein kinase 4, as a suppressor of tumor growth in SCC of the skin. We show that expression of PKK is decreased in human SCC of the skin compared with normal skin. Further, suppression of PKK in human keratinocytes leads to increased cell proliferation. The use of RNA interference to reduce PKK expression in keratinocytes leads to an increase in S phase and in proteins that promote cell cycle progression. Consistent with the results obtained from cell culture, there is a marked increased tumorigenesis after PKK knockdown in a xenotransplant model and in soft agar assays. The loss of tumor suppression involves the NF-κB and p63 pathways. NF-κB is inhibited through inhibition of inhibitor of NF-κB kinase function and there is increased nuclear TP63 activity after PKK knockdown. This study opens new avenues both in the discovery of disease pathogenesis and for potential treatments.

Objective To evaluate the requirement for protein kinase Cβ (PKCβ) in the development of lupus in mice, and to explore the potential of targeting PKCβ as a therapeutic strategy in lupus. Methods Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which represent different stages of severity in the development of lupus, were crossed with PKCβ-deficient mice. The effect of PKCβ deficiency in lupus development was analyzed. In addition, the effects of the PKCβ-specific inhibitor enzastaurin on the survival of B cells from mice with lupus and human 9G4-positive B cells as well as the in vivo effect of enzastaurin treatment on the development of lupus in Sle mice were investigated. Results In Sle mice, PKCβ deficiency abrogated lupus-associated phenotypes, including high autoantibody levels, proteinuria, and histologic features of lupus nephritis. Significant decreases in spleen size and in the peritoneal B-1 cell population, reduced numbers of activated CD4 T cells, and normalized CD4:CD8 ratios were observed. PKCβ deficiency induced an anergic B cell phenotype and preferentially inhibited autoreactive plasma cells and autoantibodies in mice with lupus. Inhibition of PKCβ enhanced apoptosis of both B cells from Sle mice and human autoreactive B cells (9G4 positive). Treatment of Sle mice with the PKCβ-specific inhibitor enzastaurin prevented the development of lupus. Conclusion This study identifies PKCβ as a central mediator of lupus pathogenesis, suggesting that PKCβ represents a promising therapeutic target for the treatment of systemic lupus erythematosus. Moreover, the results indicate the feasibility of using a PKCβ inhibitor for the treatment of lupus.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and the most common type of non-Hodgkin lymphoma. Despite recent advances in treatment, less than 50% of the patients are cured with current multiagent chemotherapy. Abnormal NF-κB activity not only contributes to tumor development but also renders cancer cells resistant to chemotherapeutic agents. Identifying and targeting signaling molecules that control NF-κB activation in cancer cells may thus yield more effective therapy for DLBCL. Here, we show that while overexpression of protein kinase C–associated kinase (PKK) activates NF-κB signaling in DLBCL cells, suppression of PKK expression inhibits NF-κB activity in these cells. In addition, we show that NF-κB activation induced by B cell–activating factor of tumor necrosis factor family (BAFF) in DLBCL cells requires PKK. Importantly, we show that knockdown of PKK impairs the survival of DLBCL cells in vitro and inhibits tumor growth of xenografted DLBCL cells in mice. Suppression of PKK expression also sensitizes DLBCL cells to treatment with chemotherapeutic agents. Together, these results indicate that PKK plays a pivotal role in the survival of human DLBCL cells and represents a potential target for DLBCL therapy.

Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

Localization of nuclear β-catenin initiates specification of vegetal fates in sea urchin embryos. We have identified SpKrl, a gene that is activated upon nuclear entry of β-catenin. SpKrl is upregulated when nuclear β-catenin activity is increased with LiCl and downregulated in embryos injected with molecules that inhibit β-catenin nuclear function. LiCl-mediated SpKrl activation is independent of protein synthesis, indicating that SpKrl is a direct target of β-catenin and TCF. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, SpKrl mRNA injection rescues some vegetal structures in β-catenin-deficient embryos. SpKrl negatively regulates expression of the animalizing transcription factor, SpSoxB1. We propose that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities.

To examine whether a BMP signaling pathway functions in specification of cell fates in sea urchin embryos, we have cloned sea urchin BMP2/4, analyzed its expression in time and space in developing embryos and assayed the developmental consequences of changing its concentration through mRNA injection experiments. These studies show that BMP4 mRNAs accumulate transiently during blastula stages, beginning around the 200-cell stage, 14 hours postfertilization. Soon after the hatching blastula stage, BMP2/4 transcripts can be detected in presumptive ectoderm, where they are enriched on the oral side. Injection of BMP2/4 mRNA at the one-cell stage causes a dose-dependent suppression of commitment of cells to vegetal fates and ectoderm differentiates almost exclusively as a squamous epithelial tissue. In contrast, NOGGIN, an antagonist of BMP2/4, enhances differentiation of endoderm, a vegetal tissue, and promotes differentiation of cells characteristic of the ciliated band, which contains neurogenic ectoderm. These findings support a model in which the balance of BMP2/4 signals produced by animal cell progeny and opposing vegetalizing signals sent during cleavage stages regulate the position of the ectoderm/ endoderm boundary. In addition, BMP2/4 levels influence the decision within ectoderm between epidermal and nonepidermal differentiation.

We have identified a Sox family transcription factor, SpSoxB1, that is asymmetrically distributed among blastomeres of the sea urchin embryo during cleavage, beginning at 4th cleavage. SpSoxB1 interacts with a cis element that is essential for transcription of SpAN, a gene that is activated cell autonomously and expressed asymmetrically along the animal-vegetal axis. In vitro translated SpSoxB1 forms a specific complex with this cis element whose mobility is identical to that formed by a protein in nuclear extracts. An anti-SpSoxB1 rabbit polyclonal antiserum specifically supershifts this DNA-protein complex and recognizes a single protein on immunoblots of nuclear proteins that comigrates with in vitro translated SpSoxB1. Developmental immunoblots of total proteins at selected early developmental stages, as well as EMSA of egg and 16-cell stage proteins, show that SpSoxB1 is present at low levels in unfertilized eggs and progressively accumulates during cleavage. SpSoxB1 maternal transcripts are uniformly distributed in the unfertilized egg and the protein accumulates to similar, high concentrations in all nuclei of 4- and 8-cell embryos. However, at fourth cleavage, the micromeres, which are partitioned by asymmetric division of the vegetal 4 blastomeres, have reduced nuclear levels of the protein, while high levels persist in their sister macromeres and in the mesomeres. During cleavage, the uniform maternal SpSoxB1 transcript distribution is replaced by a zygotic nonvegetal pattern that reinforces the asymmetric SpSoxB1 protein distribution and reflects the corresponding domain of SpAN mRNA accumulation at early blastula stage (∼150 cells). The vegetal region lacking nuclear SpSoxB1 gradually expands so that, after blastula stage, only cells in differentiating ectoderm accumulate this protein in their nuclei. The results reported here support a model in which SpSoxB1 is a major regulator of the initial phase of asymmetric transcription of SpAN in the nonvegetal domain by virtue of its distribution at 4th cleavage and is subsequently an important spatial determinant of expression in the early blastula. This factor is the earliest known spatially restricted regulator of transcription along the animal-vegetal axis of the sea urchin embryo.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell–like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell–like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.