Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling.

International audienceRecent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells

A strategy for constructing aneuploid yeast strains by transient nondisjunction of a target chromosome

<p>Abstract</p> <p>Background</p> <p>Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In <it>Saccharomyces cerevisiae</it>, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication.</p> <p>Results</p> <p>The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and <it>ura3::HIS3</it>, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene <it>TUB2</it>. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene <it>TUB1</it>, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes.</p> <p>Conclusion</p> <p>This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect.</p

The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Adverse childhood experiences and subjective cognitive decline: An analysis of the Behavioral Risk Factor Surveillance System

Background: Cognitive functioning plays a crucial role in maintaining a healthy, active, and independent lifestyle. A 2017 study found the total net cost of care for an individual with dementia was 175% more than a person without dementia 1 . With an aging population and increasing rates of dementia in the U.S., improved etiology of cognitive decline is pertinent to establishing preventative measures, and therefore slowing increasing rates. The aim of this study was to determine the association between domains of Adverse Childhood Experiences (ACEs), and subjective cognitive decline (SCD) in a representative sample of the US adult population.Methods: Data was obtained from the 2019 and 2020 Behavioral Risk Factor Surveillance Survey (N=18,096; > 45 years). ACEs were summed and categorized into 0, 1-2, and 3+ for ACE accumulation analysis. Among individuals reporting one ACE, domains of adversity (Family Mental Illness, Family Substance Abuse, Family Incarceration, Parental Divorce, Intimate Partner Violence, Emotional Abuse, Physical Abuse, and Sexual Abuse) were compared to those reporting 0 ACEs. We estimated prevalence of ACEs among individuals responding to the SCD questions within BRFSS and used multivariate logistic regression to determine the association between ACE domains and SCD.Results: Our sample included 178,441 respondents representing a population estimate of 38,215,839. Among respondents aged 45 and over, 10.14% (n = 18,096; N = 3,960,992) reported experiencing cognitive decline. Mean ACE scores among participants reporting cognitive decline were 2.61 compared to an ACE score 1.44 in participants not reporting cognitive decline, a statistically significant difference (P<.001).Compared to individuals reporting 0 ACEs, individuals reporting 1-2 ACEs were more likely to report frequently experiencing memory (OR: 1.59; 95%CI 1.43-1.76) and even greater among those reporting 3 or more ACEs (OR: 3.58; 95%CI: 3.23-3.96). Individuals reporting 3 or more ACEs were also significantly more likely to report frequent difficulties with ADLs, needing assistance with ADLs, and experiencing social limitations due to cognitive decline compared to individuals with no ACEs. Further, those with higher ACEs scores were significantly less likely to have spoken with a healthcare provider about their cognitive decline. Among individuals reporting 1 ACE of family mental illness, family substance abuse, family incarceration, emotional abuse, and physical abuse had significantly greater odds of reporting memory loss compared to individuals with no ACEs. Individuals with 1 ACE of parental divorce were less likely to get help with ADLs when needed, and individuals reporting 1 ACE of sexual abuse were significantly less likely to experience social limitations compared to those with no ACEs.Conclusions: Having multiple ACEs was significantly associated with higher odds of cognitive decline and associated limitation of social activity and inversely associated with getting help when it is needed. Further, many ACE domains were associated with SCD—a novel addition to the literature and the methodology used herein. Interventions focused on improving cognitive health and preventing cognitive decline should consider the potential role of ACEs among affected population

Association of high-sensitivity C-reactive protein with de novo major depression

Background: Although there is cross-sectional evidence that changes in the immune system contribute to the pathophysiology of depression, longitudinal data capable of elucidating cause and effect relationships are lacking. Aims: We aimed to determine whether subclinical systemic inflammation, as measured by serum high-sensitivity C-reactive protein (hsCRP) concentration, is associated with an increased risk of de novo major depressive disorder. Method: Major depressive disorder was diagnosed using a clinical interview (SCID-I/NP). This is a retrospective cohort study; from a population-based sample of 1494 randomly selected women recruited at baseline during the period 1994-7, 822 were followed for a decade and provided measures of both exposure and outcome. Of these women, 644 (aged 20-84 years) had no prior history of depression at baseline and were eligible for analysis. Results: During 5827 person-years of follow-up, 48 cases of de novo major depressive disorder were identified. The hazard ratio (HR) for depression increased by 44% for each standard deviation increase in log-transformed hsCRP (ln-hsCRP) (HR = 1.44, 95% CI 1.04-1.99), after adjusting for weight, smoking and use of non-steroidal anti-inflammatory drugs. Further adjustment for other lifestyle factors, medications and comorbidity failed to explain the observed increased risk for depression. Conclusions: Serum hsCRP is an independent risk marker for de novo major depressive disorder in women. This supports an aetiological role for inflammatory activity in the pathophysiology of depression

Breast Cancer Stem Cells Are Regulated by Mesenchymal Stem Cells through Cytokine Networks

International audienceWe have used in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSC) and bone marrow-derived mesenchymal stem cells (MSC). We show that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice, labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerated tumor growth by increasing the breast CSC population. With immunochemistry, we identified MSC-CSC niches in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow-derived MSCs may accelerate human breast tumor growth by generating cytokine networks that regulate the CSC population. Cancer Res; 71(2); 614-24. (C) 2011 AACR

&apos;&apos;Positive Examples&apos;&apos;: A Bottom-Up Approach to Identifying Best Practices in HIV Care and Treatment Based on the Experiences of Peer Educators

Abstract Literature describing the roles and activities of peers working in HIV care is limited. Evaluations of the impact of peer-based behavior-change interventions reveal mixed results, due in part to varied program aims, structure, evaluation mechanisms, and training. Peers themselves are important resources to address these concerns and lay the groundwork for developing improved programs and evaluation strategies. This qualitative article describes peer support in HIV care and treatment from the perspective of 23 HIV-positive peers across the United States. Peers reported that peer characteristics (HIV-status, common experiences, and self-care) enable them to engage clients. Peers also required flexibility to respond to client needs, and their activities spanned four types of social support: informational, emotional, instrumental, and affiliational. We recommend peer programs and evaluations accommodate the broad scope of peer work by acknowledging the need for flexibility and activities that are not always directly related to clients&apos; HIV care and treatment

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome

International audienceApplication of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts