Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

Pathways linking socioeconomic position and diabetes: The role of psychosocial factors

Despite increasing interest in the relationship between socioeconomic position (SEP) and health, there remains little understanding of the mechanisms through which SEP is related to chronic disease. This dissertation utilized data from 2,592 U.S. households in the 1995 telephone survey of the Aging, Status, and the Sense of Control study to: (1) investigate potential mediating factors in the association between educational level and prevalence of diabetes and (2) to investigate the association between the three major measures of SEP—income, education, and occupation—and the prevalence of diabetes. Regression analyses were conducted to examine the degree to which sense of personal control and social support mediate the association between level of educational attainment and diabetes and to examine the contribution of each of the SEP measures to diabetes. After adjusting for age, obesity, sex, and race, respondents with less than a high school education had greater odds of having diabetes than those with a college degree or higher level of educational attainment, although the corresponding confidence interval contained the null value (OR = 1.2, 95% CI: 0.7, 2.0). Neither sense of control nor social support significantly mediated the association between education and diabetes. However, sense of control was associated with diabetes status (OR = 0.7, 95% CI: 0.5, 1.0). Compared with income and education, employment status was the most strongly associated measure of SEP with diabetes prevalence. After adjusting for age, obesity, sex, and race, respondents who were unable to work due to disability had fourfold greater odds of having diabetes than those who were employed full time (OR = 4.0; 95% CI: 1.9, 8.3). Adding income and/or education to the model did not improve the fit. Understanding the impact of socioeconomic factors on diabetes requires consideration of multiple measures of SEP as well as the psychosocial pathways through which SEP may influence diabetes. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Evaluation of the causal pathways through which subjective norms influence mammography intention and stage of change in a national sample of women veterans

Few studies have investigated causal pathways linking psychosocial factors to each other and to screening mammography. Conflicting hypotheses exist in the theoretic literature regarding the role and importance of subjective norms, a person's perceived social pressure to perform the behavior and his/her motivation to comply. The Theory of Reasoned Action (TRA) hypothesizes that subjective norms directly affect intention; while the Transtheoretical Model (TTM) hypothesizes that attitudes mediate the influence of subjective norms on stage of change. No one has examined which hypothesis best predicts the effect of subjective norms on mammography intention and stage of change. Two statistical methods are available for testing mediation, sequential regression analysis (SRA) and latent variable structural equation modeling (LVSEM); however, software to apply LVSEM to dichotomous variables like intention has only recently become available. No one has compared the methods to determine whether or not they yield similar results for dichotomous variables. ^ Study objectives were to: (1) determine whether the effect of subjective norms on mammography intention and stage of change are mediated by pros and cons; and (2) compare mediation results from the SRA and LVSEM approaches when the outcome is dichotomous. We conducted a secondary analysis of data from a national sample of women veterans enrolled in Project H.O.M.E. (H&barbelow;ealthy O&barbelow;utlook on the M&barbelow;ammography E&barbelow;xperience), a behavioral intervention trial. ^ Results showed that the TTM model described the causal pathways better than the TRA one; however, we found support for only one of the TTM causal mechanisms. Cons was the sole mediator. The mediated effect of subjective norms on intention and stage of change by cons was very small. These findings suggest that interventionists focus their efforts on reducing negative attitudes toward mammography when resources are limited. ^ Both the SRA and LVSEM methods provided evidence for complete mediation, and the direction, magnitude, and standard errors of the parameter estimates were very similar. Because SRA parameter estimates were not biased toward the null, we can probably assume negligible measurement error in the independent and mediator variables. Simulation studies are needed to further our understanding of how these two methods perform under different data conditions. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

Evaluation of therapist and client language in Motivational Interviewing (MI) sessions: A secondary analysis of data from the Southern Methodist Alcohol Research Trial (SMART) study

In the United States, “binge” drinking among college students is an emerging public health concern due to the significant physical and psychological effects on young adults. The focus is on identifying interventions that can help decrease high-risk drinking behavior among this group of drinkers. One such intervention is Motivational interviewing (MI), a client-centered therapy that aims at resolving client ambivalence by developing discrepancy and engaging the client in change talk. Of late, there is a growing interest in determining the active ingredients that influence the alliance between the therapist and the client. This study is a secondary analysis of the data obtained from the Southern Methodist Alcohol Research Trial (SMART) project, a dismantling trial of MI and feedback among heavy drinking college students. The present project examines the relationship between therapist and client language in MI sessions on a sample of “binge” drinking college students. Of the 126 SMART tapes, 30 tapes (‘MI with feedback’ group = 15, ‘MI only’ group = 15) were randomly selected for this study. MISC 2.1, a mutually exclusive and exhaustive coding system, was used to code the audio/videotaped MI sessions. Therapist and client language were analyzed for communication characteristics. Overall, therapists adopted a MI consistent style and clients were found to engage in change talk. Counselor acceptance, empathy, spirit, and complex reflections were all significantly related to client change talk (p-values ranged from 0.001 to 0.047). Additionally, therapist ‘advice without permission’ and MI Inconsistent therapist behaviors were strongly correlated with client sustain talk (p-values ranged from 0.006 to 0.048). Simple linear regression models showed a significant correlation between MI consistent (MICO) therapist language (independent variable) and change talk (dependent variable) and MI inconsistent (MIIN) therapist language (independent variable) and sustain talk (dependent variable). The study has several limitations such as small sample size, self-selection bias, poor inter-rater reliability for the global scales and the lack of a temporal measure of therapist and client language. Future studies might consider a larger sample size to obtain more statistical power. In addition the correlation between therapist language, client language and drinking outcome needs to be explored.^

EGFR and Notch signaling pathways are regulated by distinct isoforms of Drosophila cbl

Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, the appropriate spatial and temporal down-regulation of signaling receptors is essential for normal development and homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of growth factor receptors through ubiquitin-mediated endocytosis and lysosomal degradation. Here, we report the D-cbl mutant phenotypes in the Drosophila eye. D-cbl mutants display overgrowth, inhibition of apoptosis, differentiation defects and increased ommatidial spacing. Many of these phenotypes are caused by lack of down-regulation of the Drosophila EGFR signaling. However, not all D-cbl phenotypes can be explained by inappropriate EGFR activity. We found that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. Strikingly, the long isoform, D-CblL, preferentially regulates the EGFR, whereas the short isoform, D-CblS, preferentially regulates Notch. Taken together, these data suggest that D-Cbl controls at least two signaling pathways, EGFR and Notch, through production of two alternatively spliced isoforms during development in Drosophila.^

CARMA3 serves as a critical mediator of NF-kappaB in multiple signal transduction pathways

The central dogma of molecular biology dictates that DNA is transcribed into RNA, which is later translated into protein. One of the early activators in this process is the transcription factor NF-κB. We have determined that an NF-κB inducer, CARMA3, is required for proper neural tube closure, similar to other NF-κB inducers. Using a genetic knockout of CARMA3, we demonstrated that it is required for Gαq-coupled GPCR-induced NF-κB activation. This is facilitated through a MAPK and IKK phosphorylation-independent mechanism, most likely by controlling NEMO-associated ubiquitination. We have also shown that CARMA3 is required for EGF and HRG-induced NF-κB activation. This activation requires the activity of both EGFR and HER2, as well as PKC. Again, we observed no defect in IKK phosphorylation, although we determined a clear defect in IKK activation. Finally, we have begun to determine the role of CARMA3 to both EGFR and HER2-induced tumorigenicity. By overexpressing a constitutive active mutant of HER2 in our CARMA3 WT and KO MEF cells, we have shown CARMA3 is important for HER2-driven soft agar colony growth. We have also shown that knockdown of endogenous CARMA3 in the EGFR-overexpressing A431 cell line abolishes EGF-induced NF-κB activation. These same cells have a dramatically reduced capacity to form colonies in soft agar as well. Using both mouse xenografts and a transgenic model of HER2-induced breast cancer, we have initiated studies which will help to determine the role of CARMA3 to in vivo tumorigenesis. Collectively, this work reveals novel roles for the CARMA3 protein in development, GPCR and EGFR/HER2 signaling. It also suggests that CARMA3 is involved in EGFR/HER2 mediated tumorigenesis, possibly indicating a novel therapeutic target for use in treatment of cancer. ^

The welfare state determinants of health: Implications of regimes and pathways

Background. Population health within and between nations is heavily influenced by political determinants, yet these determinants have received significantly less attention than socioeconomic factors in public health. It has been hypothesized that the welfare state, as a political variable, may play a particularly prominent role in affecting both health indicators and health disparities in developed countries. The research, however, provides conflicting evidence regarding the health impact of particular regimes over others and the mechanisms through which the welfare state can most significantly affect health.^ Objective. To perform a systematic review of the literature as a means of exploring what the current research indicates regarding the benefits or detriments of particular regimes styles and the pathways through which the welfare state can impact heath indicators and health disparities within developed countries.^ Methods. A thorough search of the EBSCO, Pubmed, Medline, Web of Science, and Scopus electronic databases was conducted and resulted in the identification of 15 studies that evaluated the association between welfare state regime and population health outcomes, and/or pathways through with the welfare state influences health. ^ Results. Social democratic countries tended to perform best when infant mortality rate (IMR) was the primary outcome of interest, whereas liberal countries performed strongly in relation to self perceived health. The results were mixed regarding welfare state effectiveness in mitigating health inequities, with Christian democratic countries performing as well as social democratic countries. In relation to welfare state pathways, public health spending and medical coverage were associated with positive health indicators. Redistributive impact of the welfare state was also consistently associated with better health outcomes while social security expenditures were not.^ Discussion/Conclusions. Studies consistently discovered a significant relationship between the welfare state and population health and/or health disparities, lending support to the hypothesis that the welfare state is, indeed, an important non-medical determinant of health. However, it is still fairly unclear which welfare state regime may be most protective for health, as results varied according to the measured health indicator. The research regarding welfare state pathways is particularly undeveloped, and does not provide much insight into the importance of in-kind service provision or cash transfers, or targeted or universal approaches to the welfare state. Suggestions to direct future research are provided.^

If we call them, will they change? Feasibility of a telephone adaptation of motivational interviewing aimed at parents to reduce television time among 2nd and 4th grade children

Television viewing is a sedentary behavior that is modifiable. Reducing media-related behaviors via parent-focused interventions may hold promise for decreasing childhood obesity. This feasibility study examines a novel parent-centered brief telephone-delivered Motivational Enhancement Interview (MEI) to encourage parents of 2nd and 4th graders to set a rules limiting TV time or remove the TV from the child's bedroom. This quasi-experimental sub-study was part of the larger CATCH: En Vivo pilot study conducted in a Hispanic population in the Texas Rio Grande Valley. Parents in the MEI condition were contacted via telephone and encouraged to meet the American Academy of Pediatrics (AAP) recommended 2 hours per day or less of TV. Unconditional logistic regression was used to analyze the data. At post-test, 85% of parents of 4th graders in the MEI group (compared to 71% at pre-test) reported having a rule limiting TV time. The adjusted odds ratio for the MEI group compared to the control group was 3.88, 95% CI (0.72-20.99). At pre-test, 63.16% of 2nd graders had a television in their bedrooms. The 2nd grade MEI intervention reduced that number to 41.03% (OR=0.25, 95%CI (0.08-0.82)). This first look at using MEI to target parents of children to modify TV behavior presents evidence on a promising strategy for modifying children's home media environment and warrants further investigation. ^

Pediatric motivational interviewing to address obesity and overweight

Not only are obesity and overweight associated with a myriad of medical and psychological concerns in childhood, but these health problems can persist even into adulthood. With the obvious need for better approaches in tackling childhood obesity and overweight and the strong evidence of motivational interviewing's effectiveness in the adult population, it is likely that an updated review will provide a more complete picture of the application of motivational interviewing in the pediatric population. The objective of this review is whether or not motivational interviewing will be instrumental in promoting factors that help in reducing the number of children who are or become overweight or obese. A literature search was completed that identified ten studies that incorporated MI as an intervention. Studies focused on either behavior health changes like increased physical activity, improved nutrition, or reducing parameters of weight like BMI or body composition. Three out of the 10 articles were still in progress and no results were provided. Five studies were able to achieve statistical significance in health benefits to control childhood overweight and obesity. Results revealed the overall feasibility of applying MI in the pediatric overweight and obese population, but the scarce number of studies limits conclusions about its efficacy. Therefore, additional scientific evidence is necessary to fully understand MI's role in affecting behavior changes in this population.^

Motivational interviewing and feedback for college drinkers: Handling missing values with complete case analysis and multiple imputation

Objective: In this secondary data analysis, three statistical methodologies were implemented to handle cases with missing data in a motivational interviewing and feedback study. The aim was to evaluate the impact that these methodologies have on the data analysis. ^ Methods: We first evaluated whether the assumption of missing completely at random held for this study. We then proceeded to conduct a secondary data analysis using a mixed linear model to handle missing data with three methodologies (a) complete case analysis, (b) multiple imputation with explicit model containing outcome variables, time, and the interaction of time and treatment, and (c) multiple imputation with explicit model containing outcome variables, time, the interaction of time and treatment, and additional covariates (e.g., age, gender, smoke, years in school, marital status, housing, race/ethnicity, and if participants play on athletic team). Several comparisons were conducted including the following ones: 1) the motivation interviewing with feedback group (MIF) vs. the assessment only group (AO), the motivation interviewing group (MIO) vs. AO, and the intervention of the feedback only group (FBO) vs. AO, 2) MIF vs. FBO, and 3) MIF vs. MIO.^ Results: We first evaluated the patterns of missingness in this study, which indicated that about 13% of participants showed monotone missing patterns, and about 3.5% showed non-monotone missing patterns. Then we evaluated the assumption of missing completely at random by Little's missing completely at random (MCAR) test, in which the Chi-Square test statistic was 167.8 with 125 degrees of freedom, and its associated p-value was p=0.006, which indicated that the data could not be assumed to be missing completely at random. After that, we compared if the three different strategies reached the same results. For the comparison between MIF and AO as well as the comparison between MIF and FBO, only the multiple imputation with additional covariates by uncongenial and congenial models reached different results. For the comparison between MIF and MIO, all the methodologies for handling missing values obtained different results. ^ Discussions: The study indicated that, first, missingness was crucial in this study. Second, to understand the assumptions of the model was important since we could not identify if the data were missing at random or missing not at random. Therefore, future researches should focus on exploring more sensitivity analyses under missing not at random assumption.^

Targeting AKT/mTOR Signaling Pathways During Murine Skin Tumor Promotion and the Impact of Dietary Energy Balance Manipulation

The prevalence of obesity has continued to rise over the last several decades in the United States lending to overall increases in risk for chronic diseases including many types of cancer. In contrast, reduction in energy consumption via calorie restriction (CR) has been shown to be a potent inhibitor of carcinogenesis across a broad range of species and tumor types. Previous data has demonstrated differential signaling through Akt and mTOR via the IGF-1R and other growth factor receptors across the diet-induced obesity (DIO)/CR spectrum. Furthermore, mTORC1 is known to be regulated directly via nutrient availability, supporting its role in the link between epithelial carcinogenesis and diet-induced obesity. In an effort to better understand the importance of mTORC1 in the context of both positive and negative energy balance during epithelial carcinogenesis, we have employed the use of specific pharmacological inhibitors, rapamycin (mTORC1 inhibitor) and metformin (AMPK activator) to target mTORC1 or various components of this pathway during skin tumor promotion. Two-stage skin carcinogenesis studies demonstrated that mTORC1 inhibition via rapamycin, metformin or combination treatments greatly inhibited skin tumor development in normal, overweight and obese mice. Furthermore, mechanisms by which these chemopreventive agents may be exerting their anti-tumor effects were explored. In addition, the effect of these compounds on the epidermal proliferative response was analyzed and drastic decreases in epidermal hyperproliferation and hyperplasia were found. Rapamycin also inhibited dermal inflammatory cell infiltration in a dose-dependent manner. Both compounds also blocked or attenuated TPA-induced signaling through epidermal mTORC1 as well as several downstream targets. In addition, inhibition of this pathway by metformin appeared to be, at least in part, dependent on AMPK activation in the skin. Overall, the data indicate that pharmacological strategies targeting this pathway offset the tumor-enhancing effects of DIO and may serve as possible CR mimetics. They suggest that mTORC1 contributes significantly to the process of skin tumor promotion, specifically during dietary energy balance effects. Exploiting the mechanistic information underlying dietary energy balance responsive pathways will help translate decades of research into effective strategies for prevention of epithelial carcinogenesis.

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^