Molecular Mechanisms Of Pressure-Stimulated Cancer Cell Signaling

ABSTRACT MOLECULAR MECHANISMS OF PRESSURE-STIMULATED CANCER CELL SIGNALING by CHRISTINA DOWNEY June 2010 Advisor: Dr. Marc Basson, MD, PhD Major: Cancer Biology Degree: Doctor of Philosophy Increased extracellular pressure stimulates cancer cell adhesion by a mechanism that is dependent upon beta-1-integrin activation, an intact cytoskeleton, and FAK and Src activation. By a different mechanism, increased extracellular pressure modulates cancer cell proliferation in a manner that is regulated by protein kinase C, but not Src or an intact cytoskeleton. Previous studies from our laboratory have shown that paxillin is a necessary mediator in the pathway by which pressure stimulates adhesion; however, it had not been determined whether paxillin acts simply as a scaffold, or participates in diverse signaling events. I therefore hypothesized that paxillin phosphorylation mediates pressure stimulated adhesion and further asked whether the independent pathway by which pressure induces proliferation is modulated by NF-kB. Finally, I postulated that increased pressures of the circulation activate cancer cells to increase adhesion, proliferation and overall tumor burden in vivo. My data illustrate that paxillin phosphorylation at tyrosines 31 and 118 is necessary for pressure stimulated adhesion, but tyrosine 81 phosphorylation is not. The phosphorylation events at tyrosine 31 and 118 form a docking site for the adaptor molecule Crk, which has a binding site for the highly phosphorylated molecule Cas. Each member of the paxillin/Crk/Cas complex is necessary for pressure stimulated adhesion. Furthermore, this complex promotes the activation of the small GTPase, Rac1. Next, I evaluated whether Rac1 was necessary for pressure induced proliferation, and found that it is not. However, it was found that this pathway is dependent upon NF-kB activation. Increased extracellular pressure increases NF-kB activation in colon, breast and prostate cancer cells. This increase is abolished in the presence of NF-kB inhibitors. Further studies showed that increased pressure activates protein kinase C alpha/beta increases IKK and IkB phosphorylation, cyclin D1 expression and increased S-phase fractions. Immunohistochemical staining for NF-kB, IkB and cyclin D1 increased in the high pressure center of human tumors and decreased toward the periphery. Finally, I determined that increased pressures of the circulation activate cancer cells in vivo by phosphorylation of beta-1-integrin, FAK and Src. Pre-treatment with pressure prior to systemic injection did not increase metastasis. However, in a local metastasis model, increased tumor burden and decreased long term survival occurred when cells were pre-treated with pressure. These increases were abolished when cells were pre-treated with FAK or Akt inhibitors prior to injection. In summary, my data suggests pressure activation of malignant cells promotes tumor development and impairs tumor free survival. Preoperative inhibition of paxillin, NF-kB, or other interventions aimed at blocking pressure-induced integrin activation may abolish this effect

Lay Theories of Health: Multidimensional Conceptualizations of What Comprises Health in Young and Middle-Aged Adults.

The present research sought to identify the content, structure, and conceptual and behavioral correlates of lay theories of health in young and middle-aged adults. Lay theories of health are the unarticulated beliefs of laypeople about what it is to be healthy. These theories were assessed in a Prestudy through open-ended survey questioning of 262 adults at four community sites, as well as nationally over the Internet. After an initial coding and judgment process by trained research assistants, 325 distinct responses to the item asking participants to describe a “very healthy person” were identified. Further judgment resulted in an item pool of 259 items to be studied in the next stage of the research. These responses were then rated on their importance to health by laypeople and by experts in separate studies (Studies 1 and 1b). Lay and expert theories about dimensions of health were compared, and some differences were revealed. Items which were rated as most important to health by laypeople (95 items) were administered to a third sample of adult laypeople over the Internet (Study 2), along with some other reliable and valid wellness measures. Participants also rated a set of five empirically-derived profiles of fictional individuals on their healthiness and unhealthiness. These profiles were comprised of items which had been rated as important to health in Study 1 (e.g., by laypeople). Ratings of these profiles showed the hypothesized pattern of increasing with higher correspondence to an “ideal” health profile. Responses to the 95 layperson-generated items were analyzed through exploratory factor analytic procedures, and five dimensions of health were identified. These were labeled Social-Emotional Health, Positive Health Practices, Absence of Illness, Absence of Stress and Anxiety, and Adequate Rest. A new measure of lay theories of health was created measuring these dimensions, as well as a summary score called Multidimensional Health. Initial validation of this measure was conducted through comparing it to the other measures of well-being administered in Study 2, and through its associations with self-reports of selected health behaviors. Findings were discussed in relation to clinical practice, research in other disciplines, and various theories of health behavior.Ph.D.PsychologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58386/1/clapan_1.pd

One-pot three-step thioconjugate addition-oxidation-Diels–Alder reactions of ethyl propiolate

Ethyl propiolate undergoes one-pot three-step thioconjugate addition-oxidation-Diels–Alder cycloaddition when treated with a variety of thiols in the presence of catalytic base, meta-chloroperbenzoic acid, lithium perchlorate, and cyclopentadiene. The reaction of S‑aryl thiols is catalyzed by trialkylamines, and the reaction of aliphatic thiols requires catalytic alkoxide base. Yields of the major diastereomer of the conveniently functionalized bicyclic products range from 47 to 81% depending upon the thiol reactant, which compares favorably to yields observed when the entire synthesis is performed step by step

One-pot synthesis of (Z)-B-sulfonyl enoates from ethyl propiolate

B-Sulfonyl enoates may be synthesized through a one-pot two-step sequence from ethyl propiolate with good to excellent selectivity for the Z isomer. Trialkylamines catalyze thioconjugate additions of aryl thiols, and alkoxides catalyze the addition of aliphatic thiols. Addition of meta-chloroperbenzoic acid (mCPBA) and LiClO4 to the reaction mixture provides rapid access to the sulfonyl enoates. Yields of the pure Z isomer range from 51 – 90%

Closest string with outliers

Background: Given n strings s1, …, sn each of length ℓ and a nonnegative integer d, the CLOSEST STRING problem asks to find a center string s such that none of the input strings has Hamming distance greater than d from s. Finding a common pattern in many – but not necessarily all – input strings is an important task that plays a role in many applications in bioinformatics. Results: Although the closest string model is robust to the oversampling of strings in the input, it is severely affected by the existence of outliers. We propose a refined model, the CLOSEST STRING WITH OUTLIERS (CSWO) problem, to overcome this limitation. This new model asks for a center string s that is within Hamming distance d to at least n – k of the n input strings, where k is a parameter describing the maximum number of outliers. A CSWO solution not only provides the center string as a representative for the set of strings but also reveals the outliers of the set. We provide fixed parameter algorithms for CSWO when d and k are parameters, for both bounded and unbounded alphabets. We also show that when the alphabet is unbounded the problem is W[1]-hard with respect to n – k, ℓ, and d. Conclusions: Our refined model abstractly models finding common patterns in several but not all input strings

The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network

Background: Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) blocking antibody, has been approved for the treatment of metastatic melanoma and induces adverse events (AE) in up to 64% of patients. Treatment algorithms for the management of common ipilimumab-induced AEs have lead to a reduction of morbidity, e.g. due to bowel perforations. However, the spectrum of less common AEs is expanding as ipilimumab is increasingly applied. Stringent recognition and management of AEs will reduce drug-induced morbidity and costs, and thus, positively impact the cost-benefit ratio of the drug. To facilitate timely identification and adequate management data on rare AEs were analyzed at 19 skin cancer centers. Methods and Findings: Patient files (n = 752) were screened for rare ipilimumab-associated AEs. A total of 120 AEs, some of which were life-threatening or even fatal, were reported and summarized by organ system describing the most instructive cases in detail. Previously unreported AEs like drug rash with eosinophilia and systemic symptoms (DRESS), granulomatous inflammation of the central nervous system, and aseptic meningitis, were documented. Obstacles included patientś delay in reporting symptoms and the differentiation of steroid-induced from ipilimumab-induced AEs under steroid treatment. Importantly, response rate was high in this patient population with tumor regression in 30.9% and a tumor control rate of 61.8% in stage IV melanoma patients despite the fact that some patients received only two of four recommended ipilimumab infusions. This suggests that ipilimumab-induced antitumor responses can have an early onset and that severe autoimmune reactions may reflect overtreatment. Conclusion: The wide spectrum of ipilimumab-induced AEs demands doctor and patient awareness to reduce morbidity and treatment costs and true ipilimumab success is dictated by both objective tumor responses and controlling severe side effects

DIAbetic macular oedema aNd diode subthreshold micropulse laser (DIAMONDS) : Ppotocol for a randomised clinical trial

Background In the UK, macular laser is the treatment of choice for people with diabetic macular oedema with central retinal subfield thickness (CST) < 400 μm, as per National Institute for Health and Care Excellence guidelines. It remains unclear whether subthreshold micropulse laser is superior and should replace standard threshold laser for the treatment of eligible patients. Methods DIAMONDS is a pragmatic, multicentre, allocation-concealed, randomised, equivalence, double-masked clinical trial that aims to determine the clinical effectiveness and cost-effectiveness of subthreshold micropulse laser compared with standard threshold laser, for the treatment of diabetic macular oedema with CST < 400 μm. The primary outcome is the mean change in best-corrected visual acuity in the study eye from baseline to month 24 post treatment. Secondary outcomes (at 24 months) include change in binocular best corrected visual acuity; CST; mean deviation of the Humphrey 10–2 visual field; change in percentage of people meeting driving standards; European Quality of Life-5 Dimensions, National Eye Institute Visual Functioning Questionnaire-25 and VisQoL scores; incremental cost per quality-adjusted life year gained; side effects; number of laser treatments and use of additional therapies. The primary statistical analysis will be per protocol rather than intention-to-treat analysis because the latter increases type I error in non-inferiority or equivalence trials. The difference between lasers for change in best-corrected visual acuity (using 95% CI) will be compared to the permitted maximum difference of five Early Treatment Diabetic Retinopathy Study (ETDRS) letters. Linear and logistic regression models will be used to compare outcomes between treatment groups. A Markov-model-based cost-utility analysis will extend beyond the trial period to estimate longer-term cost-effectiveness. Discussion This trial will determine the clinical effectiveness and cost-effectiveness of subthreshold micropulse laser, when compared with standard threshold laser, for the treatment of diabetic macular oedema, the main cause of sight loss in people with diabetes mellitus

School's out: what are urban children doing? The Summer Activity Study of Somerville Youth (SASSY)

Background: Research indicates that in the United States, children experience healthier BMI and fitness levels during school vs. summer, but research is limited. The primary goal of this pilot study was to assess where children spend their time during the months that school is not in session and to learn about the different types of activities they engage in within different care settings. A secondary goal of this pilot study was to learn what children eat during the summer months. Methods: A nine-week summer study of 57 parents of second and third grade students was conducted in an economically, racial/ethnically and linguistically diverse US urban city. Weekly telephone interviews queried time and activities spent on/in 1) the main caregiver’s care 2) someone else’s care 3) vacation 4) and camp. Activities were categorised as sedentary, light, moderate, or vigorous (0-3 scale). For each child, a mean activity level was calculated and weighted for proportion of time spent in each care situation, yielding a weighted activity index. On the last phone call, parents answered questions about their child’s diet over the summer. Two post-study focus groups were conducted to help interpret findings from the weekly activity interviews. Results: The mean activity index was 1.05 ± 0.32 and differed between gender (p = 0.07), education (p = 0.08) and primary language spoken in the household (p = 0.01). Children who spent a greater percentage of time in parent care had on average a lower activity index (β = -0.004, p = 0.01) while children who spent a greater percentage of time in camp had a higher activity index (β = 0.004, p = 0.03). When stratified into type of camp, percentage of time spent in active camp was also positively associated with mean activity index (β = 0.005, p =\u3c 0.001). With regards to diet, after adjusting for maternal education, children who attended less than five weeks of camp were four times more likely to eat their meals in front of the TV often/almost all of the time (OR = 4.0, 95%CI 1.0-16.2, p \u3c 0.06). Conclusions: Summer activities and some dietary behaviours are influenced by situation of care and sociodemographic characteristics. In particular, children who spend a greater proportion of time in structured environments appear to be more active. We believe that this pilot study is an important first step in our understanding of what children do during the summer months

Standard threshold laser versus subthreshold micropulse laser for adults with diabetic macular oedema : the DIAMONDS non-inferiority RCT

Background: The National Institute for Health and Care Excellence recommends macular laser to treat diabetic macular oedema with a central retinal subfield thickness of < 400 µm on optical coherence tomography. The DIAMONDS (DIAbetic Macular Oedema aNd Diode Subthreshold micropulse laser) trial compared standard threshold macular laser with subthreshold micropulse laser to treat diabetic macular oedema suitable for macular laser. Objectives: Determining the clinical effectiveness, safety and cost-effectiveness of subthreshold micropulse laser compared with standard threshold macular laser to treat diabetic macular oedema with a central retinal subfield thickness of < 400 µm. Design: A pragmatic, multicentre, allocation-concealed, double-masked, randomised, non-inferiority, clinical trial. Setting: Hospital eye services in the UK. Participants: Adults with diabetes and centre-involving diabetic macular oedema with a central retinal subfield thickness of  24 Early Treatment Diabetic Retinopathy Study letters (Snellen equivalent > 20/320) in one/both eyes. Interventions: Participants were randomised 1 : 1 to receive 577 nm subthreshold micropulse laser or standard threshold macular laser (e.g. argon laser, frequency-doubled neodymium-doped yttrium aluminium garnet 532 nm laser); laser treatments could be repeated as needed. Rescue therapy with intravitreal anti-vascular endothelial growth factor therapies or steroids was allowed if a loss of ≥ 10 Early Treatment Diabetic Retinopathy Study letters between visits occurred and/or central retinal subfield thickness increased to > 400 µm. Main outcome measures: The primary outcome was the mean change in best-corrected visual acuity in the study eye at 24 months (non-inferiority margin 5 Early Treatment Diabetic Retinopathy Study letters). Secondary outcomes included the mean change from baseline to 24 months in the following: binocular best-corrected visual acuity; central retinal subfield thickness; the mean deviation of the Humphrey 10–2 visual field in the study eye; the percentage of people meeting driving standards; and the EuroQol-5 Dimensions, five-level version, National Eye Institute Visual Function Questionnaire – 25 and Vision and Quality of Life Index scores. Other secondary outcomes were the cost per quality-adjusted life-years gained, adverse effects, number of laser treatments and additional rescue treatments. Results: The DIAMONDS trial recruited fully (n = 266); 87% of participants in the subthreshold micropulse laser group and 86% of participants in the standard threshold macular laser group had primary outcome data. Groups were balanced regarding baseline characteristics. Mean best-corrected visual acuity change in the study eye from baseline to month 24 was –2.43 letters (standard deviation 8.20 letters) in the subthreshold micropulse laser group and –0.45 letters (standard deviation 6.72 letters) in the standard threshold macular laser group. Subthreshold micropulse laser was deemed to be not only non-inferior but also equivalent to standard threshold macular laser as the 95% confidence interval (–3.9 to –0.04 letters) lay wholly within both the upper and lower margins of the permitted maximum difference (5 Early Treatment Diabetic Retinopathy Study letters). There was no statistically significant difference between groups in any of the secondary outcomes investigated with the exception of the number of laser treatments performed, which was slightly higher in the subthreshold micropulse laser group (mean difference 0.48, 95% confidence interval 0.18 to 0.79; p = 0.002). Base-case analysis indicated no significant difference in the cost per quality-adjusted life-years between groups. Future work: A trial in people with ≥ 400 µm diabetic macular oedema comparing anti-vascular endothelial growth factor therapy alone with anti-vascular endothelial growth factor therapy and macular laser applied at the time when central retinal subfield thickness has decreased to < 400 µm following anti-vascular endothelial growth factor injections would be of value because it could reduce the number of injections and, subsequently, costs and risks and inconvenience to patients. Limitations: The majority of participants enrolled had poorly controlled diabetes. Conclusions: Subthreshold micropulse laser was equivalent to standard threshold macular laser but required a slightly higher number of laser treatments. Trial registration: This trial is registered as EudraCT 2015-001940-12, ISRCTN17742985 and NCT03690050. Funding: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 26, No. 50. See the NIHR Journals Library website for further project information

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients