Lessons learned in the practice of community-based participatory research with community partner collaboration in study design and implementation: the community scientist model.

Engagement of community participation is an innovative driver of modern research. However, to benefit the communities being studied, it is imperative to continuously evaluate ethical considerations, the relationship dynamic between researchers and community members, and the responsiveness of research teams to the needs and preferences of communities. Northwestern University's Center for Health Equity Transformation founded a community scientist program in 2018 that implemented a study using the Community-Based Participatory Research (CBPR) model. This project is an ongoing study of heavy metal exposure by geographic location in Chicago. Community scientists from various backgrounds, communities, and organizations formed an advisory panel, partnering with the cancer research team. This commentary describes lessons learned in structuring meaningful community involvement and benefit in CBPR, with a focus on three lessons learned that relate to ethics, relationships, and responsiveness. Our findings lay new groundwork for iteratively shaping best practices in CBPR.

Systematic Patient Navigation Strategies to Scale Breast Cancer Disparity Reduction by Improved Cancer Prevention and Care Delivery Processes.

PURPOSE: Patient navigation uses trained personnel to eliminate barriers to timely care across all phases of the health care continuum, thereby reducing health disparities. However, patient navigation has yet to be systematized in implementation models to improve processes of care at scale rather than remain a band-aid approach focused solely on improving care for the individual patient. The 4R systems engineering approach (right information and right treatment to the right patient at the right time) uses project management discipline principles to develop care sequence templates that serve as patient-centered project plans guiding patients and their care team. METHODS: A case-study approach focused on the underserved patient shows how facilitators to timely breast cancer screening and care pragmatically identified as emergent data by patient navigators can be actionized by iteratively revising 4R care sequence templates to incorporate new insights as they emerge. RESULTS: Using a case study of breast cancer screening of a low-income patient, we illustrate how 4R care sequence templates can be revised to incorporate emergent facilitators to care identified through patient navigation. CONCLUSION: Use of care sequence templates can inform the care team to optimize a particular patient's care, while functioning as a learning health care system for process improvement of patient care and patient navigation scaling. A learning health care system approach that systematically integrates data patterns emerging from multiple patient navigation experiences through in-person navigators and 4R care sequence templates may improve processes of care and allow patient navigation scaling to reduce cancer disparities.

Leveraging an Implementation Science Framework to Adapt and Scale a Patient Navigator Intervention to Improve Mammography Screening Outreach in a New Community.

Helping Her Live (HHL) is a community health worker-led outreach model that navigates women from vulnerable communities to mammography screening and diagnostic follow-up. The objective of this study was to evaluate HHL implementation on the southwest side of Chicago. HHL has been implemented on the west side of Chicago since 2008, where it has increased mammogram completion and diagnostic follow-up rates among Black and Hispanic women from resource poor communities. In 2014, HHL was translated to the southwest side of Chicago; implementation success was evaluated by comparing outreach, navigation request, and mammogram completion metrics with the west side. During January 2014-December 2015, outreach was less extensive in the southwest setting (SW) compared to the benchmark west setting (W); however, the proportion of women who completed mammograms in SW was 50%, which compared favorably to the proportion observed in the benchmark setting W (42%). The distribution of insurance status and the racial and ethnic makeup of individuals met on outreach in the W and SW were significantly different (p < 0.0005). This successful expansion of HHL in terms of both geographic and demographic reach justifies further studies leveraging these results and tailoring HHL to additional underserved communities.

Improving Research Literacy in Diverse Minority Populations with a Novel Communication Tool.

Racial/ethnic minorities are underrepresented in clinical research in the USA for multifarious reasons, including barriers to effective communication between researchers and potential research participants. To address the communication barriers between researchers and potential participants, we developed a Research Literacy Support (RLS) tool. The focus of this report is to present findings from the second and third phases of development that refined and assessed usability of the RLS tool. We utilized a mixed-methods approach that entailed iterative cognitive testing with participants (N = 52) from diverse racial/ethnic backgrounds and interviews with clinical research recruiters (N = 20) to modify and refine the design and content of the RLS tool (phase 2). This was followed by assessment of the usability of the RLS tool by 100 participants (phase 3). During phase 2, participants provided feedback about layout, word choice, and comprehension of the tool. In phase 3, participants recognized that they had gained knowledge about clinical research from the RLS tool, although they still had a substantial learning gap after using the tool, indicating an opportunity for further refinement. The RLS tool may help advance health equity by addressing communication barriers that may impede minority participation in clinical research.

Protein kinase Calpha and epsilon small-molecule targeted therapeutics: a new roadmap to two Holy Grails in drug discovery?

Protein kinase (PK)Calpha and epsilon are rational targets for cancer therapy. However, targeted experimental therapeutics that inhibit PKCalpha or epsilon are unavailable. The authors established recently that covalent modification of an active-site cysteine in human PKCepsilon, Cys452, by small molecules, for example 2-mercaptoethanolamine, is necessary and sufficient to render PKCepsilon kinase-dead. Cys452 is conserved in only eleven human protein kinase genes, including PKCalpha. Therefore, the design of small molecules that bind PKC active sites with an electrophile substituent positioned proximal to the Cys452 side chain may lead to targeted therapeutics that selectively inhibit PKCepsilon, PKCalpha or other PKC isozymes.

Identification of an inactivating cysteine switch in protein kinase Cepsilon, a rational target for the design of protein kinase Cepsilon-inhibitory cancer therapeutics.

Critical roles played by some protein kinases in neoplastic transformation and progression provide a rationale for developing selective, small-molecule kinase inhibitors as antineoplastic drugs. Protein kinase Cepsilon (PKCepsilon) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKCepsilon is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKCepsilon inhibitors that block activation by DAG or compete with ATP have not yet met with success, suggesting a need for new strategies. We previously reported that cystamine and a metabolic cystine precursor inactivate PKCepsilon in cells in a thiol-reversible manner. In this report, we first determined that PKCepsilon became resistant to inactivation by disulfides when Cys452 was replaced with alanine by site-specific mutagenesis of human PKCepsilon or a constitutively active PKCepsilon mutant. These results showed that the disulfides inactivated PKCepsilon by thiol-disulfide exchange, either upon Cys452 S-thiolation or by rearrangement to an intra-protein disulfide. Mass spectrometric analysis of peptide digests of cystamine-inactivated, carbamidomethylated PKCepsilon detected a peptide S-cysteaminylated at Cys452, indicating that Cys452 S-cysteaminylation is a stable modification. Furthermore, PKCepsilon inactivation by N-ethylmaleimide was Cys452 dependent, providing corroborative evidence that PKCepsilon inhibitors can be designed by targeting Cys452 with small molecules that stably modify the residue. Cys452 is an active site residue that is conserved in only 11 human protein kinase genes. Therefore, the PKCepsilon-inactivating Cys452 switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKCepsilon.

Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission.

Cell signaling entails a host of post-translational modifications of effector-proteins. These modifications control signal transmission by regulating the activity, localization or half-life of the effector-protein. Prominent oxidative modifications induced by cell-signaling reactive oxygen species (ROS) are cysteinyl modifications such as S-nitrosylation, sulfenic acid and disulfide formation. Disulfides protect protein sulfhydryls against oxidative destruction and simultaneously influence cell signaling by engaging redox-regulatory sulfhydryls in effector-proteins. The types of disulfides implicated in signaling span (1) protein S-glutathionylation, e.g. as a novel mode of Ras activation through S-glutathionylation at Cys-118 in response to a hydrogen-peroxide burst, (2) intra-protein disulfides, e.g. in the regulation of the stability of the protein phosphatase Cdc25C by hydrogen-peroxide, (3) inter-protein disulfides, e.g. in the hydrogen peroxide-mediated inactivation of receptor protein-tyrosine phosphatase alpha (RPTPalpha) by dimerization and (4) protein S-cysteaminylation by cystamine. Cystamine is a byproduct of pantetheinase-catalyzed pantothenic acid recycling from pantetheine for biosynthesis of Coenzyme A (CoA), a ubiquitous and metabolically indispensable cofactor. Cystamine inactivates protein kinase C-epsilon (PKCepsilon), gamma-glutamylcysteine synthetase and tissue transglutaminase by S-cysteaminylation-triggered mechanisms. The importance of protein S-cysteaminylation in signal transmission in vivo is evident from the ability of cystamine administration to rescue the intestinal inflammatory-response deficit of pantetheinase knockout mice. These mice lack the predominant epithelial pantetheinase isoform and have sharply reduced levels of cystamine/cysteamine in epithelial tissues. In addition, intraperitoneal administration of cystamine significantly delays neurodegenerative pathogenesis in a Huntington's disease mouse model. Thus, cystamine may serve as a prototype for the development of novel therapeutics that target effector-proteins regulated by S-cysteaminylation.

PKC sulfhydryl targeting by disulfiram produces divergent isozymic regulatory responses that accord with the cancer preventive activity of the thiuram disulfide.

The protein kinase C (PKC) isozyme family plays key roles in cell growth regulation and influences neoplastic disease development and progression. For example, PKCepsilon is oncogenic, and PKCdelta tumor-suppressive. PKC isozymes are characterized by distinct activation mechanisms entailing phosphatidylserine-dependent cofactor binding to the regulatory domain. Evidence is now emerging that redox signaling offers another platform of PKC regulation. We have established that PKC isozymes are regulated by S-thiolation, a posttranslational modification entailing disulfide linkage of low-molecular-weight species to select protein sulfhydryls. Our recent studies demonstrate that physiologically occurring disulfides with cysteinyl constituents, e.g., cystine, regulate cellular PKC isozymes by S-thiolation-triggered mechanisms. This report shows that PKC isozymes are also molecular targets of a chemically distinct class of disulfides. Disulfiram is a thiuram disulfide with potent cancer preventive activity in in vivo models of chemical carcinogenesis. Our results indicate that PKC Sthiolation by disulfiram induces differential regulatory effects on PKC isozymes that correlate with the cancer preventive activity of the drug. The implication of these findings is that PKC-regulatory effects of thiuram disulfides may offer a useful pharmacological guide for development of disulfiram analogues with superior cancer preventive activity.

Protein kinase C-{alpha} mediates epidermal growth factor receptor transactivation in human prostate cancer cells.

Progression of human prostate cancer to a malignancy that is refractory to androgen-ablation therapy renders the disease resistant to available treatment options and accounts for the high prostate cancer mortality rate. Epidermal growth factor receptor (EGFR) expression in human prostate cancer specimens increases with disease progression to androgen-refractory prostate cancer, and experimental models implicate EGFR-dependent signaling to Erk1/2 activation in the androgen-refractory prostate cancer phenotype. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced Erk1/2 activation in human prostate cancer PC-3 cells is a paradigm of diacylglycerol-induced EGFR transactivation in androgen-independent prostate cancer. In this report, we establish an obligatory role for TPA-induced protein kinase C (PKC)-alpha activation in EGFR transactivation and signaling to Erk1/2 activation in PC-3 cells. TPA-regulated molecules include PKCs, PKDs, and Ras guanyl nucleotide-releasing proteins. The PKC-selective inhibitors GF109203X and Go6983 each blocked TPA-induced EGFR transactivation, indicating a requirement for PKC. PC-3 cells express four PKC isozymes. Prolonged bryostatin 1 treatment abrogated PKCalpha expression without altering expression levels of the other PKC isozymes. Pharmacologic PKCalpha "knockdown" abrogated TPA-induced Erk1/2 activation without affecting the EGF/EGFR-induced response, indicating that PKCalpha was required for EGFR transactivation but dispensable for signaling of ligand-activated EGFR to Erk1/2 activation. We corroborated this by showing that Go6976, which is a PKCalpha-selective inhibitor in PC-3 cells, likewise abolished TPA-induced Erk1/2 activation and did not inhibit EGF/EGFR-induced Erk1/2 activation. Go6976 had similar effects in DU145 cells, providing evidence for a common PKCalpha-dependent Erk1/2 activation mechanism in androgen-independent human prostate cancer cells of distinct genetic origin. These results constitute a rational basis for selective PKCalpha inhibition as a modality of prostate cancer therapy.

Resveratrol antagonizes EGFR-dependent Erk1/2 activation in human androgen-independent prostate cancer cells with associated isozyme-selective PKC alpha inhibition.

The development of androgen-independent prostate cancer (AI PrCa) involves constitutive Erk1/2 activation sustained by the epidermal growth factor/transforming growth factor-alpha/EGF receptor (EGF/TGFalpha/EGFR) axis and other trophic signaling mechanisms in neoplastic human prostate epithelial cells in vivo. In this report, we show that growth-inhibitory concentrations of the dietary phytochemical resveratrol suppress EGFR-dependent Erk1/2 activation pathways stimulated by EGF and phorbol ester (12- O -tetradecanoyl phorbol 13-acetate, TPA) in human AI PrCa PC-3 cells in vitro. Because protein kinase C (PKC) is the major cellular receptor for phorbol esters and taking into consideration that resveratrol is PKC-inhibitory, we investigated resveratrol effects on cellular PKC isozymes associated with the suppression of TPA-induced Erk1/2 activation. The PKC isozyme composition of PC-3 cells was defined by Western analysis of the cell lysate with a comprehensive set of isozyme-selective PKC Ab's. PC-3 cells expressed PKCalpha, epsilon, zeta, iota, and PKD (PKCmicro), as did another human AI PrCa cell line of distinct genetic origin, DU145. The effects of resveratrol on TPA-induced PKC isozyme activation were defined by monitoring PKC isozyme translocation and autophosphorylation. Under conditions where resveratrol suppressed TPA-induced Erk1/2 activation, the phytochemical produced isozyme-selective interference with TPA-induced translocation of cytosolic PKCalpha to the membrane/cytoskeleton and selectively diminished the amount of autophosphorylated PKCalpha in the membrane/cytoskeleton of the TPA-treated cells. These results demonstrate that resveratrol abrogation of a PKC-mediated Erk1/2 activation response in PC-3 cells correlates with isozyme-selective PKCalpha inhibition. The results provide evidence that resveratrol may have value as an adjuvant cancer therapeutic in advanced prostate cancer.

Cellular protein kinase C isozyme regulation by exogenously delivered physiological disulfides--implications of oxidative protein kinase C regulation to cancer prevention.

We reported previously that cystine produces regulatory responses in purified, recombinant human protein kinase C-delta (PKCdelta) and PKCepsilon via S-thiolation-triggered mechanisms that are consistent with a cancer preventive effect, i.e. stimulation of the pro-apoptotic, tumor-suppressive isozyme PKCdelta and inactivation of the growth-stimulatory, oncogenic isozyme PKCepsilon, at S-cysteinylation stoichiometries that correspond to modification of a single redox-regulatory cysteine (Cys) switch in each isozyme. In this report, we show that the oxidative regulatory responses of purified PKCdelta and PKCepsilon to cystine are recapitulated in disulfide-treated cells. We report that treatment of COS7-PKCepsilon transfectants with the cystine precursor cystine dimethyl ester (CDME) produced concentration- and time-dependent PKCepsilon inactivation that was associated with oxidative PKCepsilon modification manifested as attenuated band intensity in PKCepsilon immunoblot analyses, and that both PKCepsilon inactivation and modification were reversed by dithiothreitol (DTT) as well as by thioredoxin. We also show that CDME induced biphasic PKCdelta regulation in COS7-PKCdelta transfectants, with DTT-irreversible PKCdelta stimulation at low and DTT-reversible PKCdelta inactivation at high CDME concentrations. The degrees of PKCdelta versus PKCepsilon inactivation by CDME treatment of COS7-PKC transfectants indicate substantial resistance of PKCdelta to inactivation. The PKCdelta stimulatory response in COS7-PKCdelta cells was triggered only by the disulfide agent and not by its reduced thiol counterpart, providing evidence for an oxidative mechanism. Also paralleling the oxidative stimulation of purified PKCdelta by cystine, the stimulation of PKCdelta elicited by CDME treatment of cells involved a stable structural change, which was evident from the stability of the stimulated form of PKCdelta to immunoprecipitation. Demonstration of oxidative regulation of cellular PKCdelta and PKCepsilon by disulfides in this report provides evidence that redox-regulatory sites in PKCdelta and PKCepsilon may offer novel targets for development of cancer preventive or therapeutic agents that selectively inactivate PKCepsilon or stimulate PKCdelta.

Resveratrol: a candidate nutritional substance for prostate cancer prevention.

The dietary stilbene resveratrol is a major constituent of a variety of edible plant products, including grapes and peanuts. Resveratrol has been identified as an excellent candidate cancer chemopreventive, based on its safety and efficacy in animal models of carcinogenesis. Resveratrol is a prototype of a plethora of bioactive polyphenols in the food supply that has just begun to be mined for cancer preventive agents. For example, polyphenolic grapeseed fractions were shown recently to potently antagonize chemical carcinogenesis. Taking into consideration that the identification of resveratrol as a cancer preventive agent is largely owed to its high abundance in nature (e.g., it accounts for 5-10% of the grapeskin biomass), it is logical to expect that naturally occurring stilbenes that are superior to resveratrol in their cancer preventive properties await identification. Thus, resveratrol may represent the tip of the iceberg of a broad class of stilbene and related polyphenolic natural products that include safe and highly effective agents for cancer prevention. We hypothesize that resveratrol may be especially suitable as a lead agent for prostate cancer prevention given its ability to: 1) inhibit each stage of multistage carcinogenesis, 2) scavenge incipient populations of androgen-dependent prostate cancer cells through androgen receptor antagonism, and 3) scavenge incipient populations of androgen-independent prostate cancer cells by short-circuiting the epidermal growth factor-receptor (EGFR)-dependent autocrine loops in the cancer cells.

PKC isozyme S-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKC delta and inactivates the oncogenic isozyme PKC epsilon.

Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth and survival. We recently reported that diamide, a diazene carbonyl derivative which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, induces potent GSH-dependent inactivation of several PKC isozymes, including the oncogenic isozyme PKC epsilon, via S-glutathiolation. PKC delta, a pro-apoptotic isozyme, was distinguished by its resistance to inactivation. In this report, we show that PKC-regulatory S-thiolation modifications produced by physiological disulfides elicit opposing effects on PKC delta and PKC epsilon activity. We report that PKC delta is stimulated 2.0-2.5 fold by GSSG, (Cys-Gly)(2) and cystine, under conditions where PKC gamma and PKC epsilon are fully inactivated by cystine, and PKC alpha activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKC delta stimulation and PKC gamma and PKC epsilon inactivation, indicative of oxidative regulation. By analyzing DTT-reversible isozyme radiolabeling by [(35)S]cystine, we demonstrate that PKC gamma, PKC delta and PKC epsilon are each [(35)S] S-cysteinylated in association with the concentration-dependent regulation of isozyme activity by cystine. The restricted reactivity of cystine, together with the effects of DTT and thioredoxin on cystine-induced PKC isozyme regulation reported here, indicate that the cystine-induced PKC-regulatory effects entail isozyme S-cysteinylation. We recently hypothesized that antagonism of tumor promotion/progression by small cellular thiols may involve PKC regulation via oxidant-induced S-thiolation reactions with PKC isozymes. The findings of cystine-induced PKC isozyme regulation by S-cysteinylation reported here offer correlative support to the hypothetical model. Thus, PKC delta, a potent antagonist of DMBA-TPA-induced tumor promotion/progression in mouse skin, is stimulated by S-cysteinylation, PKC epsilon, an important mediator of the tumor promotion/progression response, is inactivated by S-cysteinylation, and PKC alpha, which is not influential in DMBA-TPA-induced tumor promotion/progression, is not regulated by cystine. Furthermore, PKC gamma has oncogenic activity, and S-cysteinylation inactivated PKC gamma and PKC epsilon similarly. These findings provide evidence that S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for development of novel cancer preventive agents.

Induction of tumor-reactive CTL by C-side chain variants of the CTL epitope HER-2/neu protooncogene (369-377) selected by molecular modeling of the peptide: HLA-A2 complex.

To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser(5) (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-gamma production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75(+) TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.

Treatment with HER-2 phosphorylation agonists enhance tumor ability to stimulate epitope specific CTL in vitro.

The transmembrane (TM) receptor encoded by the HER-2 proto-oncogene (HER-2) is amplified in several types of human carcinomas and premalignant states and provides an important target for cancer therapy. While overexpression of HER-2 should lead to increased CTL epitope formation due to the attendant increase in higher protein turnover, breast tumors are poor stimulators of CTL. In this report, we show that treatment of SKBR3.A2 tumor cells with HER-2 receptor agonists (EGF and NDF) enhanced tumor ability to activate CTL from tumor associated lymphocytes (TAL) and from T cells from peripheral blood in vitro. The enhanced ability of tumor cells to stimulate CTL was paralleled by tyrosine phosphorylation of HER-2, and its oligo-ubiquitination compared with control untreated, or TPA-treated tumor cells. Our results demonstrate that HER-2 ligands used at concentrations which induce tyrosine phosphorylation but not downregulation of the receptor can be used to enhance the ability of tumor cells to activate CTL. This may have implications for overcoming Ag ignorance and tolerance in human cancers.