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PKB/Akt kinase localization and role in stemness maintenance in cancer
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancer incidence rates have increased over the last decade. Currently available therapies are only moderately effective in targeting cancer cells. Established cancer treatment protocols fail to eliminate populations of cancer stem cells (CSCs), which develop resistance against the chemotherapeutic drugs and lead to cancer recurrence. Therefore, understanding the mechanisms by which CSCs resist drugs and identifying molecular markers are both necessary to further improve prognosis and to develop new treatment strategies. Increased protein kinase B/Akt1 gene expression and/or activity have been found increased in majority of cancer types. Akt1 is a key player in PI3K-AktmTOR pathway that is vital for cell survival, proliferation, migration, invasion, metastasis, angiogenesis and apoptosis. In this study, we investigated a series of novel markers to improve the characterization of CSCs, with particular focus the roles of Akt in CSC maintenance and the regulatory role of micro-RNA (miR) in cancer cells. While utilizing in breast cancer cells as models, we found that luminescent conjugated oligothiophenes (LCOs), p-HTMI and p-HTAA have the potential to differentially stain various subpopulations of cancer cells, presumably also CSCs among breast cancer cells. However, further studies are needed to confirm these results. Additionally, when we investigated the effect of Akt intracellular compartmentalization on CSC development, the results revealed that nuclear Akt enhances CSC proliferation (ALDH +/High CD44+/High/CD24-/Low) and clonogenicity, which was counter examined and confirmed by using the Aktspecific inibitor triciribine. Furthermore, while investigating the impact of Akt on miR regulation in cancer cells, we found that Akt overexpression decreased.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 60 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1515
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:liu:diva-127477DOI: 10.3384/diss.diva-127477ISBN: 978-91-7685-806-6 (print)OAI: oai:DiVA.org:liu-127477DiVA: diva2:923959
Public defence
2016-06-01, Belladonna, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2016-04-27 Created: 2016-04-27 Last updated: 2016-04-27Bibliographically approved
List of papers
1. Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
Open this publication in new window or tab >>Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
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2014 (English)In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 85A, no 7, 628-635 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescent compounds capable of staining cells selectively without affecting their viability are gaining importance in biology and medicine. Recently, a new family of optical dyes, denoted luminescent conjugated oligothiophenes (LCOs), has emerged as an interesting class of highly emissive molecules for studying various biological phenomena. Properly functionalized LCOs have been utilized for selective identification of disease-associated protein aggregates and for selective detection of distinct cells. Herein, we present data on differential staining of various cell types, including cancer cells. The differential staining observed with newly developed pentameric LCOs is attributed to distinct side chain functionalities along the thiophene backbone. Employing flow cytometry and fluorescence microscopy we examined a library of LCOs for stainability of a variety of cell lines. Among tested dyes we found promising candidates that showed strong or moderate capability to stain cells to different extent, depending on target cells. Hence, LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells, whereas LCOs with other amino acid side chains along the backbone showed a complete lack of staining for the cells included in the study. Furthermore, for p-HTMI,a LCO functionalized with methylated imidazole moieties, the staining was dependent on the p53 status of the cells, indicating that the molecular target for the dye is a cellular component regulated by p53. We foresee that functionalized LCOs will serve as a new class of optical ligands for fluorescent classification of cells and expand the toolbox of reagents for fluorescent live imaging of different cells.

Place, publisher, year, edition, pages
John Wiley & Sons, 2014
Keyword
cancer stem cells; luminescent conjugated oligothiophenes; fluorescent probes
National Category
Clinical Medicine Chemical Sciences Medical Biotechnology Computer and Information Science
Identifiers
urn:nbn:se:liu:diva-109171 (URN)10.1002/cyto.a.22437 (DOI)000338007700010 ()24500794 (PubMedID)
Available from: 2014-08-12 Created: 2014-08-11 Last updated: 2017-12-05Bibliographically approved
2. Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21(Waf1/Cip1) and p27(kip1)
Open this publication in new window or tab >>Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21(Waf1/Cip1) and p27(kip1)
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2015 (English)In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 14, no 13, 2109-2120 p.Article in journal (Refereed) Published
Abstract [en]

Cancer stem-like cells (CSCs) are a rare subpopulation of cancer cells capable of propagating the disease and causing cancer recurrence. In this study, we found that the cellular localization of PKB/Akt kinase affects the maintenance of CSCs. When Akt tagged with nuclear localization signal (Akt-NLS) was overexpressed in SKBR3 and MDA-MB468 cells, these cells showed a 10-15% increase in the number of cells with CSCs enhanced ALDH activity and demonstrated a CD44(+High)/CD24(-Low) phenotype. This effect was completely reversed in the presence of Akt-specific inhibitor, triciribine. Furthermore, cells overexpressing Akt or Akt-NLS were less likely to be in G0/G1 phase of the cell cycle by inactivating p21(Waf1/Cip1) and exhibited increased clonogenicity and proliferation as assayed by colony-forming assay (mammosphere formation). Thus, our data emphasize the importance the intracellular localization of Akt has on stemness in human breast cancer cells. It also indicates a new robust way for improving the enrichment and culture of CSCs for experimental purposes. Hence, it allows for the development of simpler protocols to study stemness, clonogenic potency, and screening of new chemotherapeutic agents that preferentially target cancer stem cells. Summary: The presented data, (i) shows new, stemness-promoting role of nuclear Akt/PKB kinase, (ii) it underlines the effects of nuclear Akt on cell cycle regulation, and finally (iii) it suggests new ways to study cancer stem-like cells.

Place, publisher, year, edition, pages
Taylor and Francis: STM, Behavioural Science and Public Health Titles, 2015
Keyword
Akt-NLS; cancer stem-like cells; mTOR; PI3K; stemness
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-120274 (URN)10.1080/15384101.2015.1041692 (DOI)000356959800021 ()26030190 (PubMedID)
Note

Funding Agencies|Linkoping University; Integrative Regenerative Medicine Center (IGEN); VR-NanoVision [K2012-99X-22325-01-5]; Cancerfonden [2013/391]; Canadian Breast Cancer Foundation (CBCF); Natural Sciences and Engineering Research Council of Canada (NSERC); [BK/265/RAU1/2014/t.10]

Available from: 2015-07-24 Created: 2015-07-24 Last updated: 2017-12-04
3. Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins
Open this publication in new window or tab >>Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins
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2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 15, 20953-20965 p.Article in journal (Refereed) Published
Abstract [en]

Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway.

Place, publisher, year, edition, pages
Impact Journals, LLC, 2016
Keyword
AKT; PI3K; PTEN; mTOR; miR301
National Category
Cell and Molecular Biology Cell Biology Cancer and Oncology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-127476 (URN)10.18632/oncotarget.7996 (DOI)000375804000140 ()26967567 (PubMedID)
Note

Funding agencies: Cancerfonden [2013/391]; GeCONiI [POIG.02.03.01-24-099/13]

Available from: 2016-04-27 Created: 2016-04-27 Last updated: 2017-11-30Bibliographically approved

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