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.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods - flow cytometry, confocal imaging, teratoma formation and transcriptional profiling - is also described. The newly generated lines express markers of embryonic stem cells - Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 - while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

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.

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.

BACKGROUND: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. METHODS:We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. RESULTS:Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. CONCLUSION:Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.

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.

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-kappa B and HIF-1 alpha in the nucleus. Enhanced binding of NF-kappa B and HIF-1 alpha 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-kappa B and HIF-1 alpha, and suppression of ROS diminishes these effects. The effect of ROS on NF-kappa B and HIF-1 alpha 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-kappa B results from phosphorylation-induced degradation of I kappa B alpha, whereas HIF-1 alpha up-regulation is NF-kappa B-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.

Abstract Pancreatic cancer (PC) remains a therapeutic challenge with a rising incidence and unabated mortality. Gemcitabine (GEM), the current standard of care drug, is largely ineffective due to chemoresistance, thus underscoring the need for a refined understanding of the molecular mechanisms that drive drug-resistant nature of this malignancy. Recently, we have shown that CXCL12/CXCR4 signaling axis is involved in conferring chemoresistance to PC cells. Here, we explored the effect of GEM on this resistance mechanism and its functional consequences. Our data demonstrated that GEM upregulated CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time- dependent manner. GEM-induced CXCR4 expression was dependent on generation of reactive oxygen species (ROS) as it was abrogated upon pre-treatment of PC cells with radical scavenger N-acetyl-L-cysteine (NAC). CXCR4 upregulation by GEM correlated with time-dependent accumulation of p65/NF-κB and HIF-1α in the nucleus. An enhanced binding of p65/NF-κB and HIF-1α to the CXCR4 promoter was observed in chromatin immunoprecipitation assay in GEM-treated PC cells, while their silencing by RNA interference suppressed GEM-induced CXCR4 expression. ROS induction upon GEM treatment preceded the nuclear accumulation of p65/NF-κB and HIF-1α and suppression of ROS diminished these effects. Effect of ROS on p65/NF-κB and HIF-1α was mediated through activation of ERK1/2 and Akt and their pharmacological inhibition also suppressed GEM-induced CXCR4 upregulation. Lastly, our data demonstrated that GEM-treated PC cells were more motile and exhibited significantly greater invasiveness against a CXCL12 gradient. Altogether, these findings reinforces the role of CXCL12/CXCR4 signaling in GEM resistance and point toward an undesired effect of chemotherapy that is potentiation of invasion and spread of resistant tumor cells. Citation Format: Sumit Arora, Arun Bhardwaj, Seema Singh, Sanjeev K. Srivastava, Steven McClellan, William E. Grizzle, Laurie B. Owen, Ajay P. Singh. An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through upregulation of CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2013-4794

Abstract Pancreatic cancer (PC) is a disease of insidious progression. In most cases, it is diagnosed late at a stage when it is either locally advanced or has already metastasized to distant sites, resulting in a poor prognosis of the patients. Therefore, there is an urgent need to identify novel molecular targets regulating PC progression and metastasis, so that an effective treatment strategy can be developed against this devastating disease. In this study, we investigated the role of Myb in pancreatic cancer pathogenesis. Myb/c-Myb, a cellular progenitor of v-Myb oncogenes, is amplified in a sub-set of PC and encodes for a transcription factor. Myb confers its oncogenic activity by regulating the expression of several target genes. To examine the pathological significance of Myb in pancreatic cancer, we silenced its expression in Myb-overexpressing pancreatic cancer cell lines (Panc1 and MiaPaCa), and ectopically overexpressed it in a poorly-tumorigenic low Myb-expressing cell line (BxPC3). Our data showed that short-hairpin RNA (shRNA)-mediated stable silencing of Myb in Panc1 and MiaPaCa cells led to diminished growth and clonogenic ability as compared to their respective scrambled-transfected control cells. Furthermore, Myb-silenced Panc1 and MiaPaCa cells also exhibited reduced, motility and invasion as compared to their respective controls. Accordingly, enhanced growth, clonogenicity, motility and invasion were observed in Myb-overexpressing BXPC3 cells as compared to vector only-transfected cells. Immunoblot analyses demonstrated altered expression of cell cycle- and apoptosis- associated proteins in Myb-silenced and overexpressing pancreatic cancer cells. Moreover, loss of mesenchymal and gain of epithelial markers was observed in Myb-silenced Panc1 and MiaPaCa cells, whereas Myb overexpressing BXPC3 cells exhibited an opposite pattern. These changes were also associated with actin reorganization, thus suggesting a role of Myb in epithelial to mesenchymal transition (EMT) of PC cells. In vivo studies in orthotopic mouse model of pancreatic cancer demonstrated decreased tumor growth and metastasis of the Myb- silenced MiaPaCa cells, while an increase was observed in Myb-overexpressing BXPC3 cells as compared to their respective control cells. Altogether, our studies provide the first experimental evidence for a functional role of Myb in progression and metastasis of pancreatic cancer. Citation Format: Sanjeev K. Srivastava, Seema Singh, Arun Bhardwaj, Sumit Arora, Steven McClellan, William E. Grizzle, Ajay P. Singh. Myb contributes to pancreatic cancer growth and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2013-3882

e13566 Background: It is well known that most cancer cells display a Warburg effect, a state of active glycolysis with lactate production under aerobic conditions. NF-κB has been shown to play a role in modulating cancer metabolism and control the balance between glycolysis and mitochondrial respiration. We have previously shown that panepoxydone (PP), a compound isolated from an edible mushroom, interferes with NF-κB mediated signal transduction. Additionally, we have shown that PP decreases cell viability and proliferation in breast cancer cells. To further characterize the antitumor effect of PP, here we have evaluated it ability to modulate cancer metabolism. Methods: The metabolic action of PP was evaluated in estrogen receptor positive (MCF-7) and triple negative (MDAMB-231) breast cancer cell lines. Cytotoxicity was determined by the cell titer glow assay and expressed as IC50. Metabolic activity, expressed as basal oxygen consumption rate (OCR, an indicator of oxidative phosphorylation) and extracellular acidification rate (ECAR, an indicator of lactate acid production), was measured in real-time using the XF24 Extracellular Flux Analyzer. Mitochondrial membrane potential (MMP) alteration was determined through flow cytometry and LDH-A expression was analyzed via western blot. Results: Significant antitumor activity with PP was seen in both MCF-7 (IC50 5 µM) and MDAMB-231 cells (IC50 15 µM). This correlated to a significant dose-dependent reduction in OCAR in MCF-7 (2-fold, p<0.05) and MDAMB-231 cells (3.5-fold, p<0.01). However, while a decrease in ECAR (1.5-fold, p<0.05), along with decreased LDHA expression was noted in the 231 cells (3.5-fold, p<0.01), this was not seen with the MCF-7 cells. A similar trend was noted with increased damaged mitochondria noted in 231-cells, but again no significant difference in MCF-7 cells. Conclusions: Taken together, these results indicate that PP has the potential to modulate metabolism in breast cancer cells, especially triple negative cells, as noted by its ability to inhibit glycolysis. The inhibition of glycolysis has become an important strategy in treating cancer and development of novel glycolytic inhibitors, such as PP, warrants its further investigation in breast cancer treatment.

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.

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/beta-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.

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.

We explored the nature of the tumor-initiating cell in osteosarcoma, a bone malignancy that predominately occurs in children. Previously, we observed expression of Oct-4, an embryonal transcriptional regulator, in osteosarcoma cell cultures and tissues. To examine the relationship between Oct-4 and tumorigenesis, cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter driving a green fluorescent protein (GFP) reporter to generate the transgenic line OS521Oct-4p. In culture, only approximately 24% of the OS521Oct-4p cells were capable of activating the transgenic Oct-4 promoter; yet, xenograft tumors generated in NOD/SCID mice contained approximately 67% GFP(+) cells, which selectively expressed the mesenchymal stem cell-associated surface antigens CD105 and ICAM-1. Comparison of the tumor-forming capacity of GFP-enriched and GFP-depleted cell fractions revealed that the GFP-enriched fractions were at least 100-fold more tumorigenic, capable of forming tumors at doses of <300 cells, and formed metastases in the lung. Clonal populations derived from a single Oct-4/GFP(+) cell were capable of forming tumors heterogeneous for Oct-4/GFP expression. These data are consistent with the cancer stem cell model of tumorigenesis in osteosarcoma and implicate a functional link between the capacity to activate an exogenous Oct-4 promoter and tumor formation. This osteosarcoma tumor-initiating cell appears highly prolific and constitutes a majority of the cell population in a primary xenograft tumor, which may provide a biological basis for the particular virulence of this type of cancer.