Lowered expression of tumor suppressor candidate MYO1C stimulates cell proliferation, suppresses cell adhesion and activates AKT

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of wellstratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition.Royal Physiographic Society in Lund (Nilsson-Ehle Foundation) with grant numbers 30928, 32705 and 36388: KV. Wilhelm and Martina Lundgren Foundation: KV, AB. Assar Gabrielsson Research Foundation for Clinical Cancer Research with grant numbers FB11-15, FB12-26, FB13-05, FB14-46 and FB15-45: KV. Sahlgrenska University Hospital Foundation with grant number 8181: KV. The Knowledge Foundation with grant number HOÈ G12, 20120311: AB.http://www.plosone.orgam2016Physiolog

An investigation into the effects of ex vivo antioxidant treatment on the regenerative potential of mesenchymal stem cells following prolonged exposure to a pathological microenvironment associated with diabetes mellitus in vivo

Thesis (PhD)--Stellenbosch University, 2019.ENGLISH ABSTRACT: Obesity-associated type-2 diabetes mellitus (T2DM) is a multifactorial disease that causes severe comorbidities such as non-healing wounds. Non-healing diabetic wounds affect 15-25% of all diabetic patients and are responsible for nearly 50% of all diabetes-related hospital admissions. Mesenchymal stem cell (MSC) therapy is a promising therapeutic option, as MSCs can ‘sense’ the clinical status of the wound and restore the micro-environment through paracrine signalling to promote regeneration. However, the pathological nature of the niche micro-environment does limit the use of autologous MSC therapy in diabetic patients, since prolonged exposure of endogenous MSCs to the diabetic environment in vivo reduces their ability to respond to environmental cues. Thus, the advancement of autologous cell therapy depends on (a) a better understanding of how the pathogenesis of T2DM affects the multifunctional properties of MSCs, and (b) the development of new strategies to restore the function of these impaired MSCs before they are used for transplantation. This study investigated whether ex vivo antioxidant [N-acetylcysteine (7.5 mM NAC) and ascorbic acid 2-phosphate (0.6 mM AAP)] treatment could restore the paracrine responsiveness, growth rate, migration ability and viability of impaired diabetic MSCs, and, if so, whether this restored state could be maintained in the presence of diabetic wound fluid (DWF). Bone marrow-derived MSCs were isolated from eight-week-old wild-type C57BL/6J mice (healthy control: MSCWT) (n = 24) and obese diabetic B6.Cg-Lepob/J mice (impaired/dysfunctional: MSCob) (n = 24). The ex vivo treatment groups (MSCWT vs MSCob) were (a) no treatment (baseline phenotype), (b) DWF-stimulated (baseline response), (c) antioxidant-preconditioned (preconditioned phenotype), and (d) antioxidant-preconditioned with subsequent DWF stimulation (preconditioned response). For these ex vivo experiments, DWF was harvested over a period of 28 days from bilateral, dorsal, full-thickness excisional wounds created on obese diabetic mice (B6.Cg-Lepob/J) (n = 7). The optimum concentration of antioxidants was determined using a dose-response experiment in immortalised C3H10/T1/2 cells. This study demonstrated that the expansion of primary MSCs (MSCWT and MSCob) in the presence of antioxidants improved the ex vivo viability of cells and had a protective effect against the toxicity of DWF. The paracrine responsiveness of MSCWT and MSCob (with and without antioxidant preconditioning) was furthermore determined at both the molecular level (mRNA expression of 84 cytokines and receptors, qPCR microarray) and protein level (23-plex bead-array Luminex assay). At baseline, 31 genes were overexpressed (more than twofold) and 39 genes were under-expressed (more than twofold) in MSCob versus MSCWT. In conditioned medium, significant baseline differences (p < 0.05) were detected for two pro-inflammatory cytokines (TNFα and IFNγ), four chemokines (KC, G-CSF, Eotaxin and MCP1) and one anti-inflammatory cytokine (IL10). Following DWF stimulation, significant differences (p < 0.05) were detected in the secretion of two chemokines (GM-CSF and Eotaxin), three pro-inflammatory cytokines (TNFα, IFNγ and IL9) and four anti-inflammatory cytokines (IL10, IL4, IL13 and IL3). Antioxidant preconditioning significantly dampened the excessive TNFα response observed in MSCob and improved the secretion of IL10. This suggests that combined ex vivo treatment of autologous MSCs with NAC and AAP could potentially be an effective strategy to restore the paracrine function of impaired diabetic MSCs before transplantation. However, despite improved viability and a restored paracrine response, antioxidant preconditioning could not rescue the proliferation and migration capacity of severely impaired diabetic MSCs.AFRIKAANSE OPSOMMING: Obesiteitsverwante tipe 2-diabetes mellitus (T2DM) is ’n komplekse siekte wat verskeie komorbiditeite veroorsaak, waaronder wonde wat sukkel om te genees. Sowat 15-25% van alle diabetespasiënte het las van sulke wonde, en bykans 50% van alle diabetesverwante hospitaalopnames kan hieraan toegeskryf word. Mesenchimale stamsel- (MSS-)terapie is ’n belowende behandelingsmoontlikheid omdat MSS’e die kliniese status van ’n wond kan ‘aanvoel’ en die mikro-omgewing deur parakriene seine kan herstel om regenerasie te bevorder. Die patologiese aard van die nis-mikro-omgewing beperk egter die gebruik van outoloë MSS-terapie by diabetespasiënte omdat verlengde blootstelling van endogene MSS’e aan die diabetiese omgewing in vivo die selle se vermoë aantas om op omgewingseine te reageer. Daarom berus die verdere ontwikkeling van outoloë selterapie op (a) ’n beter begrip van hoe die patogenese van T2DM die multifunksionele eienskappe van MSS’e beïnvloed, en (b) die ontwikkeling van nuwe strategieë om die funksie van hierdie aangetaste MSS’e te herstel voordat dit vir oorplanting gebruik word. Hierdie studie ondersoek of ex vivo-antioksidant- [N-asetielsisteïen (7.5 mM NAC) en askorbiensuur2-fosfaat-behandeling (0.6 mM AAP)] die parakriene responsiwiteit, groeitempo, migrasievermoë en lewensvatbaarheid van aangetaste diabetiese MSS’e kan herstel, en, indien wel, of hierdie herstelde toestand in die teenwoordigheid van diabetiese wondvog (DWV) gehandhaaf kan word. Beenmurgafkomstige MSS’e is op die ouderdom van agt weke geïsoleer by wildetipe-C57BL/6Jmuise (gesonde kontrole: MSCWT) (n = 24) en vetsugtige diabetiese B6.Cg-Lepob/J-muise (aangetas/disfunksioneel: MSCob) (n = 24). Die ex vivo-behandelingsgroepe (MSCWT teenoor MSCo b) het bestaan uit (a) geen behandeling (basislynfenotipe), (b) DWV-gestimuleer (basislynreaksie), (c) vooraf met antioksidante gekondisioneer (voorafgekondisioneerde fenotipe), en (d) vooraf met antioksidante gekondisioneer, met daaropvolgende DWV-stimulering (voorafgekondisioneerde reaksie). DWV vir hierdie ex vivo-proefnemings is oor ’n tydperk van 28 dae bekom uit bilaterale, dorsale eksisiewonde van volle dikte wat op diabetiese muise (B6.Cg-Lepob/J) (n = 7) aangebring is. Die optimale konsentrasie antioksidante is met behulp van ’n dosisreaksieproefneming met geïmmortaliseerde C3H10/T1/2-selle bepaal. Hierdie studie toon dat die uitbreiding van primêre MSS’e (MSCWT en MSCob) in die teenwoordigheid van antioksidante die ex vivo-lewensvatbaarheid van selle verbeter en ’n beskermende uitwerking teen die toksisiteit van DWV het. Daarbenewens is die parakriene responsiwiteit van MSCWT en MSCob (met en sonder voorafkondisionering met antioksidante) op molekulêre vlak (mRNAuitdrukking van 84 sitokiene en reseptors, qPCR-mikrorangskikking) sowel as proteïenvlak bereken (Luminex-essai, 23-pleks, kraalrangskikking). Op die basislyn is ooruitdrukking van 31 gene (meer as tweevoudig) en onderuitdrukking van 39 gene (meer as tweevoudig) by MSCob teenoor MSCWT opgemerk. In gekondisioneerde media is beduidende basislynverskille (p < 0.05) opgemerk vir twee pro-inflammatoriese sitokiene (TNFα en IFNγ), vier chemokiene (KC, G-CSF, Eotaxin en MCP1) en een anti-inflammatoriese sitokien (IL10). Ná DWV-stimulering is beduidende verskille (p < 0.05) opgemerk in die uitskeiding van twee chemokiene (GM-CSF en Eotaxin), drie pro-inflammatoriese sitokiene (TNFα, IFNγ en IL9) en vier anti-inflammatoriese sitokiene (IL10, IL4, IL13 en IL3). Voorafkondisionering met antioksidante het die waargenome TNFα-oorreaksie by MSCob aansienlik gedemp en die uitskeiding van IL10 verbeter. Dít dui daarop dat gekombineerde ex vivo-behandeling van outoloë MSS’e met NAC en AAP moontlik ’n doeltreffende strategie kan wees om die parakriene funksie van aangetaste MSS’e voor oorplanting te herstel. Ondanks beter lewensvatbaarheid en ’n herstelde parakriene reaksie, kon voorafkondisionering met antioksidante egter nie die proliferasie- en migrasievermoë van ernstig aangetaste diabetiese MSS’e red nie.Doctorat

Protein expression analysis of PI3K/AKT pathway components in cells expressing INPP5K and MYO1C

In an Experimental Rat model for endometrial carcinoma (EC) a minimal region of recurrent deletion/allelic loss at the neighborhood of the Tp53 gene has been identified. A similar observation of deletion at the homologous position on human chromosome 17 unassociated with TP53 mutation has been reported in several human cancer types. Thus an important tumor suppressor activity located close to, but distinct of TP53 is suggested. Detailed molecular analysis of this candidate region in a tumor model suggested Myo1c (myosin 1C) and Inpp5k (inositol polyphosphate-5-phosphatase K), also known as Skip (skeletal muscle and kidney enriched inositol polyphosphate phosphatase), as the best candidates. These two genes are suggested to be involved in glucose metabolism through PI3K/AKT signaling and neither of them has earlier been reported as a tumor suppressor gene. The present work aimed to investigate the potential correlation of MYO1C and/or INPP5K proteins with components of PI3K/AKT signaling pathway involved in cell growth and survival. Cells were transfected with increasing amounts of MYO1C- or INPP5K- gene expression constructs and protein extracts of the cells were subjected to Western Blot analysis for 13 important components of the signaling pathway: p110β\α\δ, p85, pAkt308&amp;473, 14-3-3β, PTEN, Akt, pErk, Erk, Ras, p4EBP1 and 4EBP1. The analysis showed dose-dependent changes in the expression levels of several of these proteins, and the observed changes for the most part were directed towards negative regulation of cell proliferation and survival. The presented data further extended the initial hypothesis for potential tumor suppressor activities of MYO1C and INPP5K proteins through PI3K/AKT pathway

Recovery from radiation-induced damage to growth plates involves functional compensation

Background: Children receiving radiotherapy during cancer treatment are highly susceptible to side-effects including short stature, irregular body proportions and spinal curvature. One reason radiotherapy impairs bone growth is that radiation directly damages cells that are responsible for bone elongation: growth plate chondrocytes. Purpose: After irradiation, bone growth can continue to a limited extent, but the underlying mechanisms of this recovery process are poorly understood. We aimed to characterize the effects of radiation on the growth plate and reveal the cellular recovery processes. Methods: The left proximal tibia of one month-old mice was irradiated(x-ray) dorso-ventrally. The effects of radiation were characterized by measuring bone lengths and conducting immunofluorescence. Recovery was analyzed using clonal genetic tracing, imaged with confocal microscopy. Results: We first conducted an irradiation-dose-response study up to 15Gy. In our model, a single dose of 10Gy focal irradiation (with biological effective dose of 38.57Gy, equivalent to 12 fractions of 2Gy) was the lowest dose that significantly reduced bone length, fourteen days after irradiation (irradiated tibia were 96.3% the length of the contralateral control, n=5, p= 0.0036). We then used clonal genetic tracing with Col2CreERT:R26R-Confetti mice to visualize the clonal recovery one month after irradiation, and made two inter-connected observations: (i) radiation dose-dependently prevented chondrocytes from further dividing, thus reducing the number of clonal-columns (Fig.1, arrowheads), (ii) some chondrocytes dose-dependently produced an increased number of columns (Fig.1, asterisk); of the clones that did produce columns, individual clones produced a mean of 4.85 columns in the irradiated side versus 2.08 columns in the contralateral control (p&lt;0.0001, unpaired t-test, n= minimum of 17 clones pooled from 4 mice). Conclusion: After radiation-induced damage, some growth plate chondrocytes can functionally compensate for the damaged cells and produce more than twice the expected number of columns. Ethical permission: All experiments were approved by the Swedish board of agriculture

Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT.

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition

Protein levels of MYO1C in endometrial hyperplasia and endometrial adenocarcinomas of stage I-III.

<p>a) Representative images for the IHC staining of MYO1C in tissue samples of endometrial carcinoma with low, medium and high MYUO1C protein levels. b) Significant association was found between MYO1C protein level and tumor grade (<i>P</i> = 0.035, linear-by-linear association in the SPSS chi-square test). The proportion of samples with high MYO1C protein level was greatest in hyperplasias (Hyp) and smallest in stage III tumors, whereas the proportion of tumors with low abundance of MYOC1 was smallest in hyperplasias and highest in stage III tumors. Pairwise differences between sample groups were not significant. c) When the samples were regrouped as tumors and hyperplasia, the Fisher’s exact test showed significant difference in MYO1C expression between these two groups of samples (<i>P</i> = 0.0303).</p

siRNA-knockdown of <i>MYO1C</i> enhanced viability.

<p>(a) Immunoblot of MYO1C protein expression in MCF10A cells transfected with <i>MYO1C</i>-siRNA and negative control (scrambled siRNA) to the final concentration of 10nM at 24, 48, 72 and 96 hours post-transfection. The image is a representative from at least three independent experiments. (b) Histogram showing percentages of relative cell viability of MCF10A cells transfected with <i>MYO1C</i>-siRNA and scrambled siRNA to the final concentration of 10nM. Significant increase in the number of live cells was observed at 48, 72 and 96 hours post-transfection compared to cells transfected with the scrambled siRNA. Each bar represents mean value ± <i>SEM</i> of all repeats (at least three independent experiments, each in quadruplicates). A Student’s <i>t</i>-test was used to compare the differences in mean value, ****<i>P</i>-value < 0.001 versus cells treated with scrambled siRNA as well as untreated cells (time point zero).</p

Decrease in cell migration and adhesion of MCF10A cells after knockdown of <i>MYO1C</i>.

<p>(a) The expression levels of <i>MYO1C</i> in the MCF10A cells subjected to 20 nM siRNA transfection were verified with RT-PCR. (b) Geometry on gap immediately after ripping of the stopper (I) and geometry on gap of the control (scrambled siRNA) (II), and <i>MYO1C</i>-siRNA transfected (III) after 24-hour incubation were shown. The white circles show the area of the geometry on gap at each measurement. (c) The analyses of the geometry on gap closure (migration assay) of MCF10A cells transfected with <i>MYO1C</i>-specific siRNA was evaluated after ripping off cell seeding stopper followed by 24-hour incubation in comparison to the geometry of control cells. (d) Immunoblot of MYO1C protein levels in MCF10A cells transiently transfected with different concentrations of <i>MYO1C</i>-siRNA (1, 3, 5 and 10 nM) and control (mock transfection) at 48 hours post-transfection. (e) Histogram showing percentages of relative cell index of MCF10A cells transfected with <i>MYO1C</i>-siRNA and control cells (received mock transfection). A dose-dependent decrease in relative cell index response to MYO1C depletion was observed reaching to a significant value with concentrations of 5 and 10 nM of <i>MYO1C</i>-siRNA at 48 hours post-transfection compared to control cells (mock transfection). Images shown in (b) and (d) are representatives from three and six independent experiments, respectively. Each bar in (a), (c) and (e) represents the mean value ± <i>SEM</i> from three and six independent experiments, respectively. A Student’s <i>t</i>-test was used to compare the differences in mean value, *<i>P</i>-value < 0.05, ***<i>P</i>-value < 0.005 and ****<i>P</i>-value < 0.001 versus control cells.</p

List of primary antibodies and the optimized dilution.

<p>List of primary antibodies and the optimized dilution.</p

Over-expression of <i>MYO1C</i> significantly decreased viability of HEK-293 cells.

<p>(a) Immunoblot of MYO1C proteins in HEK-293 cells shows increased expression in the cells transfected with different amounts of <i>MYO1C</i>-construct at 48 hours post-transfection in comparison with the cells transfected with corresponding empty plasmid, serving as control. The image is a representative from at least three independent experiments. (b) Dose-response effect of over-expression of MYO1C protein on cell proliferation was observed at 48 hours post-transfection in comparison with cells transfected with empty vector. Each bar represents mean value ± <i>SEM</i> of all repeats, a Student’s <i>t</i>-test was used to compare the differences in mean values, *<i>P</i>-value < 0.05 versus the corresponding empty plasmid. (c) Immunoblot of MYO1C proteins in HEK-293 cells shows increased expression in <i>MYO1C</i>-transfected cells at 24, 48, 72 and 96 hours post-transfection. The image is a representative from at least three independent experiments. (d) Relative number of living cells was reduced in <i>MYO1C</i>-transfected cells. Significant decline was observed at 48, 72 and 96 hours post-transfection compared to cells transfected with the empty vector. Each bar represents mean value ± <i>SEM</i> of all repeats (at least three independent experiments, each in quadruplicates). A Student’s <i>t</i>-test was used to compare the differences in mean values, ****<i>P</i>-value < 0.001 versus the corresponding empty plasmid.</p