reprogramming-primary-amniotic-fluid-membrane-cells-to-pluripotency

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

University of South Alabama

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G₁ phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.

Honokiol arrests cell cycle, induces apoptosis, and potentiates the cytotoxic effect of gemcitabine in human pancreatic cancer cells.

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

Myb overexpression overrides androgen depletion-induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance.

Chemoresistance &egrave; un ostacolo importante nel trattamento del cancro. I nostri studi precedenti hanno dimostrato che miR-125b gioca un ruolo importante nella chemoresistance. Qui riportiamo un nuovo meccanismo che up-regolazione del miR-125b attraverso segnalazione Wnt da lumaca arricchisce le cellule staminali tumorali. Sovraespressione di lumaca aumenta drammaticamente l&#39;espressione di miR-125b attraverso l&#39;asse di lumaca-attivato Wnt/&beta;-catenina/TCF4. Lumaca conferisce chemoresistance da reprimere Bak1 attraverso up-regolazione del miR-125b. Ripristinare l&#39;espressione di Bak1 o lesive miR-125b re-sensitizes lumaca-esprimendo le cellule tumorali a Taxol, indicando che miR-125b &egrave; critico in chemoresistance lumaca-indotta. Inoltre, la sovraespressione del miR-125b aumenta significativamente la popolazione di cellule staminali del cancro (CD24-CD44 +), mentre la deplezione del miR-125b o salvataggio dell&#39;espressione di Bak1 aumenta la popolazione delle cellule staminali (CD24 + CD44 +) nelle cellule con sovraesprimono relativa lumaca. Questi risultati supportano fortemente che funzioni miR-125b come un mediatore chiave nel cancro lumaca-indotta delle cellule staminali arricchimento e chemoresistance. Questo nuovo meccanismo per la propagazione di lumaca-indotta delle cellule staminali e chemoresistance pu&ograve; avere implicazioni importanti nello sviluppo di strategie per superare la resistenza delle cellule del cancro alla chemioterapia.

miR-125b funzioni come un mediatore chiave per la propagazione di lumaca-indotta delle cellule staminali e chemoresistance.

Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/Î²-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

miR-125b functions as a key mediator for snail-induced stem cell propagation and chemoresistance.

Chimior&eacute;sistance est un obstacle majeur dans le traitement du cancer. Nos &eacute;tudes ant&eacute;rieures ont montr&eacute; que miR-125 b joue un r&ocirc;le important dans la chimior&eacute;sistance. Nous pr&eacute;sentons un nouveau m&eacute;canisme de r&eacute;gulation que de miR-125 b par le biais de signalisation Wnt par escargot enrichit les cellules souches canc&eacute;reuses. La surexpression de l&#39;escargot augmente de fa&ccedil;on spectaculaire l&#39;expression de miR-125 b par l&#39;axe de l&#39;escargot-activ&eacute; Wnt/&beta;-cat&eacute;nine/TCF4. Escargot conf&egrave;re chimior&eacute;sistance en r&eacute;primant Bak1 gr&acirc;ce &agrave; une r&eacute;gulation de miR-125 b. Restaurer l&#39;expression des Bak1 ou appauvrissant miR-125 b re-sensitizes exprimant l&#39;escargot des cellules canc&eacute;reuses au Taxol, qui indique que miR-125 b est essentielle &agrave; la chimior&eacute;sistance induite par l&#39;escargot. En outre, la surexpression de miR-125 b augmente de mani&egrave;re significative la population de cellules souches du cancer (CD24-CD44 +), tandis que l&#39;appauvrissement de la couche de miR-125 b ou le sauvetage de l&#39;expression de Bak1 augmente la population non-cellules souches (CD24 + CD44 +) dans les cellules surexprimant-escargot. Ces r&eacute;sultats appuient fortement cette fonctions miR-125 b m&eacute;diateur cl&eacute; dans l&#39;enrichissement de cellules souches du cancer induite par l&#39;escargot et la chimior&eacute;sistance. Ce nouveau m&eacute;canisme pour la propagation de l&#39;escargot-induite de cellules de tige et chimior&eacute;sistance peut avoir des implications importantes dans l&#39;&eacute;laboration de strat&eacute;gies pour surmonter la r&eacute;sistance de la cellule du cancer &agrave; la chimioth&eacute;rapie.

miR-125 b fonctionne comme un m&eacute;diateur cl&eacute; pour la propagation de l&#39;escargot-induite de cellules souches et la chimior&eacute;sistance.

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through reactive oxygen species-dependent, nuclear factor κB- and hypoxia-inducible factor 1α-mediated up-regulation of CXCR4.

Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines.

Panepoxydone targets NF-kB and FOXM1 to inhibit proliferation, induce apoptosis and reverse epithelial to mesenchymal transition in breast cancer.

Identification of novel molecular targets and understanding the mechanisms underlying the aggressive nature of pancreatic cancer (PC) remain prime focus areas of research. Here, we investigated the expression and pathobiological significance of p21-activated kinase 4 (PAK4), a gene that was earlier shown to be amplified in a sub-set of PC. Our data demonstrate PAK4 overexpression in PC tissues and cell lines with little or no expression in the normal pancreas. PAK4 silencing in two PC cell lines, MiaPaCa and T3M4, by RNA interference causes suppression of growth and clonogenic ability due to decreased cell cycle progression and apoptosis-resistance. PAK4-silenced PC cells exhibit altered expression of proliferation- and survival-associated proteins. Moreover, we observe decreased nuclear accumulation and transcriptional activity of NF-κB in PAK4-silenced PC cells associated with stabilization of its inhibitory protein, IκBα. Transfection of PAK4-silenced PC cells with constitutively-active mutant of IKKβ, an upstream kinase of IκBα, leads to restoration of NF-κB activity and PC cell growth. Furthermore, we show that PAK4-induced NF-κB activity is mediated through activation and concerted action of ERK and Akt kinases. Together, these findings suggest that PAK4 is a regulator of NF-κB pathway in PC cells and can serve as a novel target for therapy.

p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway.

Emerging data highlight the significance of chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) signaling axis in the chemoresistance of several malignancies, including prostate cancer (PCa); however, underlying mechanisms remain largely elusive. Here, we demonstrate that CXCL12 treatment rescues the PCa cells from docetaxel (DTX)-induced toxicity by overriding its effect on cell cycle (G2/M phase arrest). We further demonstrate that the chemoprotective effect of CXCL12 is abolished upon pharmacological inhibition or RNA interference-mediated silencing of CXCR4. Moreover, microtubule stabilization caused by DTX is suppressed in CXCL12-stimulated PCa cells as revealed by immunofluorescence and immunoblot analyses. The effect of CXCL12 on microtubule stabilization is abrogated when PCa cells are pre-treated with a CXCR4 antagonist. In additional studies, we show that the chemoprotective action of CXCL12/CXCR4 signaling is mediated by p21-activated kinase 4 (PAK4)-dependent activation of Lim domain kinase 1 (LIMK1), and inhibition of either PAK4 or LIMK1 leads to re-sensitization of PCa cells to DTX-induced tubulin polymerization and cellular toxicity even in the presence of CXCL12. Altogether, our findings uncover a novel mechanism underlying CXCL12/CXCR4 signaling-induced PCa chemoresistance and suggest that targeting of this signaling axis or its downstream effector pathway could lead to therapeutic enhancement of DTX.

CXCL12/CXCR4 signaling counteracts docetaxel-induced microtubule stabilization via p21-activated kinase 4-dependent activation of LIM domain kinase 1.

Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technology that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers associated with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examined by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.

mRNA detection in living cells: A next generation cancer stem cell identification technique.

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24(+)/CD44(+)/EpCAM(+)) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24(-)/CD44(-)/EpCAM(-)) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemosensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling.

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α.

Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.

Article	Year
Honokiol arrests cell cycle, induces apoptosis, and potentiates the cytotoxic effect of gemcitabine in human pancreatic cancer cells. PloS one\| PubMed ID: 21720559	2011
Myb overexpression overrides androgen depletion-induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resist... Carcinogenesis\| PubMed ID: 22431717	2012
miR-125b functions as a key mediator for snail-induced stem cell propagation and chemoresistance. The Journal of biological chemistry\| PubMed ID: 23255607	2013
An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through reactive oxygen species-dependent, nuclear factor κB- and hypoxia-inducible factor 1α-me... The Journal of biological chemistry\| PubMed ID: 23740244	2013
Panepoxydone targets NF-kB and FOXM1 to inhibit proliferation, induce apoptosis and reverse epithelial to mesenchymal transition in breast cancer. PloS one\| PubMed ID: 24896091	2014
p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway. Oncotarget\| PubMed ID: 25238288	2014
CXCL12/CXCR4 signaling counteracts docetaxel-induced microtubule stabilization via p21-activated kinase 4-dependent activation of LIM domain kinase 1. Oncotarget\| PubMed ID: 25359780	2014
mRNA detection in living cells: A next generation cancer stem cell identification technique. Methods (San Diego, Calif.)\| PubMed ID: 25920950	2015
p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling. Cancer letters\| PubMed ID: 26546043	2016
Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions. Cell cycle (Georgetown, Tex.)\| PubMed ID: 26654216	2016
Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α. Cancer research\| PubMed ID: 27197253	2016
Induced pluripotent stem cells derived from human amnion in chemically defined conditions. Cell cycle (Georgetown, Tex.)\| PubMed ID: 29143560	2017

Steven McClellan

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