The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

Cellular mechanisms of operant and classical conditioning

The ability to associate a predictive stimulus with a subsequent salient event (i.e., classical conditioning) and the ability to associate an expressed behavior with the consequences (i.e., operant conditioning) allow for a predictive understanding of a changing environment. Although they are operationally distinct, there has been considerable debate whether at some fundamental level classical and operant conditioning are mechanistically distinct or similar. Feeding behavior of Aplysia (i.e., biting) was chosen as the model system and was successfully conditioned with appetitive forms of both operant and classical conditioning. The neuronal circuitry responsible for feeding is well understood and is suitable for cellular analyses, thus providing for a mechanistic comparison between these two forms of associative learning. ^ Neuron B51 is part of the feeding circuitry of Aplysia and is critical for the expression of ingestive behaviors. B51 also is a locus of plasticity following both operant and classical conditioning. Both in vivo and in vitro operant conditioning increased the input resistance and the excitability of B51. No pairing-specific changes in the input resistance were observed following both in vivo and in vitro classical conditioning. However, classical conditioning decreased the excitability of B51. Thus, both operant and classical conditioning modified the threshold level for activation of neuron B51, but in opposite directions, revealing key differences in the cellular mechanisms underlying these two forms of associative learning. ^ Next, the cellular mechanisms underlying operant conditioning were investigated in more detail using a single-cell analogue. The single-cell analogue successfully recapitulated the previous in vivo and in vitro operant conditioning results by increasing the input resistance and the excitability of B51. Both PKA and PKC were necessary for operant conditioning. Dopamine appears to be the transmitter mediating the reinforcement signal in this form of conditioning. A D1 dopamine receptor antibody revealed that the D1receptor localizes to the axon hillock, which is also the region that gives the strongest response when iontophoresing dopamine. ^ The studies presented herein, thus, provide for a greater understanding of the mechanisms underlying both of these forms of associative learning and demonstrate that they likely operate through distinct cellular mechanisms. ^

The p120-catenin/Kaiso signaling pathway

The canonical and non-canonical Wnt signaling pathways appear to interact with one another as a network in development, or when hyper-activated, in the progression of disease. A much studied key mediator of the canonical Wnt pathway, β-catenin, is characterized by a central armadillo-repeat domain that engages in multiple protein-protein interactions, such as those with cadherins functioning at cell-cell contact regions. In the nucleus, β-catenin forms a complex with the repressor TCF/LEF, promoting the activation of genes participating in processes such as proliferation, differentiation and stem cell survival. Somewhat similarly, the p120-catenin binds the distinct transcriptional repressor Kaiso, relieving Kaiso-mediated repression to promote gene activation. Here, employing Xenopus laevis, I report upon both downstream and upstream aspects of the p120-catenin/Kaiso pathway which was previously poorly understood. I first show that Kaiso, a BTB/POZ zinc-finger family member, directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1 and c-Myc) in conjunction with TCF. Depletion or dominant-negative inhibition of xKaiso results in Siamois de-repression, while xKaiso over-expression induces additional Siamois repression through recruitment of N-CoR co-repressor and chromatin modifications. Functional interdependencies are further corroborated by the capacity of Kaiso to suppress β-catenin-induced axis duplication. Thus, my work inter-relates the p120-catenin/Kaiso and β-catenin/TCF pathways at the level of specific gene promoters important in development and cancer progression. Regarding upstream aspects of the p120-catenin/Kaiso pathway, I collaboratively identified p120 in association with Frodo, a protein previously identified as a component of the canonical (β-catenin dependent) Wnt pathway. I determined that canonical Wnt signals result in Frodo-mediated stabilization of p120-catenin, resulting in the sequestration of Kaiso to the cytoplasm and thereby the activation (relief of repression) of gene targets. Developmental evidence supporting this view included findings that Frodo has the capacity to partially rescue Kaiso over-expression phenotypes in early Xenopus embryos. Taken together, my studies point to the convergence of p120-catenin/Kaiso and β-catenin/TCF signaling pathways at the level of gene transcription as well as at more upstream points during vertebrate development. ^

Identification of micro-RNAs targeting genes of PI3K/AKT pathway in ovarian cancer

Ovarian cancer is the leading cause of cancer-related death for females due to lack of specific early detection method. It is of great interest to find molecular-based biomarkers which are sensitive and specific to ovarian cancer for early diagnosis, prognosis and therapeutics. miRNAs have been proposed to be potential biomarkers that could be used in cancer prevention and therapeutics. The current study analyzed the miRNA and mRNA expression data extracted from the Cancer Genome Atlas (TCGA) database. Using simple linear regression and multiple regression models, we found 71 miRNA-mRNA pairs which were negatively associated between 56 miRNAs and 24 genes of PI3K/AKT pathway. Among these miRNA and mRNA target pairs, 9 of them were in agreement with the predictions from the most commonly used target prediction programs including miRGen, miRDB, miRTarbase and miR2Disease. These shared miRNA-mRNA pairs were considered to be the most potential genes that were involved in ovarian cancer. Furthermore, 4 of the 9 target genes encode cell cycle or apoptosis related proteins including Cyclin D1, p21, FOXO1 and Bcl2, suggesting that their regulator miRNAs including miR-16, miR-96 and miR-21 most likely played important roles in promoting tumor growth through dysregulated cell cycle or apoptosis. miR-96 was also found to directly target IRS-1. In addition, the results showed that miR-17 and miR-9 may be involved in ovarian cancer through targeting JAK1. This study might provide evidence for using miRNA or miRNA profile as biomarker.^

Prediction of sepsis in the burn intensive care unit patient

Sepsis is a significant cause for multiple organ failure and death in the burn patient, yet identification in this population is confounded by chronic hypermetabolism and impaired immune function. The purpose of this study was twofold: 1) determine the ability of the systemic inflammatory response syndrome (SIRS) and American Burn Association (ABA) criteria to predict sepsis in the burn patient; and 2) develop a model representing the best combination of clinical predictors associated with sepsis in the same population. A retrospective, case-controlled, within-patient comparison of burn patients admitted to a single intensive care unit (ICU) was conducted for the period January 2005 to September 2010. Blood culture results were paired with clinical condition: "positive-sick"; "negative-sick", and "screening-not sick". Data were collected for the 72 hours prior to each blood culture. The most significant predictors were evaluated using logistic regression, Generalized Estimating Equations (GEE) and ROC area under the curve (AUC) analyses to assess model predictive ability. Bootstrapping methods were employed to evaluate potential model over-fitting. Fifty-nine subjects were included, representing 177 culture periods. SIRS criteria were not found to be associated with culture type, with an average of 98% of subjects meeting criteria in the 3 days prior. ABA sepsis criteria were significantly different among culture type only on the day prior (p = 0.004). The variables identified for the model included: heart rate>130 beats/min, mean blood pressure<60 mmHg, base deficit<-6 mEq/L, temperature>36°C, use of vasoactive medications, and glucose>150 mg/d1. The model was significant in predicting "positive culture-sick" and sepsis state, with AUC of 0.775 (p < 0.001) and 0.714 (p < .001), respectively; comparatively, the ABA criteria AUC was 0.619 (p = 0.028) and 0.597 (p = .035), respectively. SIRS criteria are not appropriate for identifying sepsis in the burn population. The ABA criteria perform better, but only for the day prior to positive blood culture results. The time period useful to diagnose sepsis using clinical criteria may be limited to 24 hours. A combination of predictors is superior to individual variable trends, yet algorithms or computer support will be necessary for the clinician to find such models useful. ^

Effects of thymus size and involution on the contribution of recent thymic emigrants to the peripheral T cell pool

The contribution of recent thymic emigrants (RTEs) to the peripheral naïve T cell population is necessary to maintain diversity of the T cell receptor (TCR) repertoire and produce immune responses against newly encountered antigens. The thymus involutes with age, after irradiation or chemotherapy, and due to severe viral infections. Thymus involution results in decreased thymopoiesis and RTE output leading to a reduced diversity of peripheral T cells. This increases susceptibility to disease and impairs immune responsiveness to vaccines. Therefore, studies aimed at maintaining or regenerating thymic function are integral for maintaining and restoring peripheral TCR diversity. Mice that express a K5.CyclinD1 transgene expression have a severely hyperplastic thymus that fails to undergo involution. Both thymocyte and TEC development appear normal in these mice. We have used the K5.CyclinD1 transgenic model to test the hypothesis that preventing thymus involution will sustain RTE output and incorporation into the peripheral T cell pool to prevent naïve T cell depletion with age. The K5.CyclinD1 transgene was crossed to the RAG2p-GFP transgenic model so that RTEs could be tracked by the intensity of the GFP signal. The frequency and number of RTEs in naïve CD4 splenic T cells was analyzed at monthly intervals to 5 months of age. Using this double transgenic approach, we determined that preventing thymus involution does maintain or enhance the number of RTEs in the peripheral T cell pool before and after thymus involution.

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^

Critical determinants of estrogen receptor -mediated agonist activity

Exogenous ligands that bind to the estrogen receptor (ER) exhibit unique pharmacologies distinct from that observed with the endogenous hormone, 17β-estradiol (ED. Differential activity among ER ligands has been observed at the level of receptor binding, promoter interaction and transcriptional activation. Furthermore, xenoestrogens can display tissue-specific agonist activity on the cellular level, functioning as an agonist in one tissue and as an antagonist in another. That the same ligand, functioning through the same receptor, can produce differing agonist responses on the cellular level indicates that there are tissue-specific determinants of agonist activity. In these studies critical molecular determinants of agonist activity were characterized for several cell types. In the normal and neoplastic myometrium a proliferative response was dependent upon activation of AF2 of the ER, functioning as a determinant of agonism in this cell type. Progesterone receptor (PR) ligands transdominantly suppressed ER-mediated transcription and proliferation in uterine leiomyoma cells, indicating that ER/PR cross-talk can modulate agonist activity in a myometrial cell background. In the breast, the agonist response to ER ligands was investigated by employing a functional genomics approach to generate gene expression profiles. Treatment of breast cancer cells with the selective estrogen receptor modulator tamoxifen largely recapitulated the expression profile induced by treatment with the agonist E2, despite the well-characterized antiproliferative effects produced by tamoxifen in this cell type. While the expression of many genes involved in regulating cell cycle progression, including fos, myc, cdc25a, stk15 and cyclin A, were induced by both E2 and tamoxifen in breast cells, treatment with the agonist E2 specifically induced the expression of cyclin D1, fra-1 , and uracil DNA glycosylase. These results suggest that the inability of tamoxifen to transactivate expression of only a few key genes, functioning as cellular gatekeepers, prevent tamoxifen-treated breast cells from entering the cell cycle. Thus, the expression of these agonist-specific marker genes is a potential determinant of agonist activity at the cellular level in the breast. Collectively, studies in the breast and uterine myometrium have identified several mechanisms whereby ER ligands modulate ER-mediated signaling and provide insights into the biology of tissue-specific agonist activity in hormone-responsive tissues. ^

Roles of p38 mitogen-activated protein kinase pathway in the regulation of SOX9 activity and chondrogenesis

Sox9 is a master transcription factor in chondrocyte differentiation. Several lines of evidence suggest that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in chondrocyte differentiation. In the present study, we examined the roles of p38 in the regulation of SOX9 activity and chondrogenesis. ^ COS7 cells were transfected with a SOX9 expression vector and 4x48-p89, a luciferase construction harboring four tandem copies of a SOX9-dependent 48-bp enhancer in Col2a1. Coexpression of MKK6EE, a constitutively active mutant of MKK6, a MAPKK that specifically activates p38, further increased the activity of the SOX9-dependent 48-bp enhancer about 5-fold, and SOX9 protein levels were not increased under these conditions. This increase in enhancer activity was not observed in a mutant enhancer construct harboring mutations that abolish SOX9 binding. These data strongly suggested that activation of the p38 pathway results in increased activity of SOX9. In addition, the increase of the activity of the SOX9-dependent 48-bp enhancer by MKK6EE was also observed in primary chondrocytes, and this increase was abolished by coexpression of a p38 phosphatase, MKP5, and p38 specific inhibitors. Furthermore, treatment of primary chondrocytes with p38 inhibitors decreased the expression of Col2a1, a downstream target of Sox9, without affecting Sox9 RNA levels, further supporting the hypothesis that p38 plays a role in regulating Sox9 activity in chondrocytes. ^ To further study the role of the p38 MAPK pathway in chondrogenesis, we generated transgenic mice that express MKK6EE in chondrocytes under the control of the Col2a1 promoter/intron regulatory sequences. These mice showed a dwarf phenotype characterized by reduced chondrocyte proliferation and a delay in the formation of primary and secondary ossification centers. Histological analysis using in situ hybridization showed reduced expression of Indian hedgehog, PTH/PTHrP receptor, cyclin D1 and increased expression of p21. In addition, consistent with the notion that Sox9 activity was increased in these mice, transgenic mice that express MKK6EE in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in chondrocyte differentiation and suggests that Sox9 is a downstream target of the p38 MAPK pathway. ^