Kyung-Mi Bae

Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

PURPOSE We identified a discrete population of stem cell-like tumor cells expressing 5 essential transcription factors required to reprogram pluripotency in prostate tumor cell lines and primary prostate cancer tissue. MATERIALS AND METHODS DU145 and PC3 human prostate cancer cell lines (ATCC), tumor tissue from patients with prostate cancer and normal prostate tissue were evaluated for the reprogramming factors OCT3/4 (Cell Signaling Technology), SOX2, Klf4 (Santa Cruz Biotechnology, Santa Cruz, California), Nanog (BioLegend) and c-Myc (Cell Signaling) by semiquantitative reverse transcriptase-polymerase chain reaction, histological and immunohistochemical analysis. Stem cell-like tumor cells were enriched by flow cytometric cell sorting using E-cadherin (R&D Systems) as a surface marker, and soft agar, spheroid and tumorigenicity assays to confirm cancer stem cell-like characteristics. RESULTS mRNA expression of transcription factors OCT3/4 and SOX2 highly correlated in primary prostate tumor tissue samples. The number of OCT3/4 or SOX2 expressing cells was significantly increased in prostate cancer tissue compared to that in normal prostate or benign prostate hyperplasia tissue (p <0.05). When isolated from the DU145 and PC3 prostate cancer cell lines by flow cytometry, stem cell-like tumor cells expressing high OCT3/4 and SOX2 levels showed high tumorigenicity in immunodeficient mice. In vivo growth of the parental DU145 and PC3 prostate cancer cell lines was inhibited by short hairpin RNA knockdown of OCT3/4 or SOX2. CONCLUSIONS Data suggest that prostate tumor cells expressing pluripotent stem cell transcription factors are highly tumorigenic. Identifying such cells and their importance in prostate cancer growth could provide opportunities for novel targeting strategies for prostate cancer therapy.

Protein Kinase Cε Is Overexpressed in Primary Human Non–Small Cell Lung Cancers and Functionally Required for Proliferation of Non–Small Cell Lung Cancer Cells in a p21/Cip1-Dependent Manner

The protein kinase C (PKC) family of proteins plays important roles in growth regulation and is implicated in tumorigenesis. It has become clear that the role of PKC in tumorigenesis is cell context dependent and/or isoform specific. In this study, we showed for the first time by immunohistochemistry that overexpression of PKC epsilon was detected in the vast majority (>90%) of primary human non-small cell lung cancers (NSCLC) compared with normal lung epithelium. Inhibition of the PKC epsilon pathway using a kinase-inactive, dominant-negative PKC epsilon, PKC epsilon(KR), led to a significant inhibition of proliferation and anchorage-independent growth of human NSCLC cells in a p53-independent manner. This was accompanied by a specific induction of the cyclin-dependent kinase (cdk) inhibitor p21/Cip1 but not p27/Kip1. In response to serum stimulation, PKC epsilon(KR)-expressing cells showed a prolonged G(1)-S transition and delayed and reduced activation of cdk2 complexes, which was likely attributed to the increased binding of p21/Cip1 to cdk2. Furthermore, inhibition of PKC epsilon function either by expressing PKC epsilon(KR) or by small interfering RNA (siRNA)-mediated gene knockdown resulted in c-Myc down-regulation, which, in turn, regulated p21/Cip1 expression. Knockdown of PKC epsilon or c-Myc expression using siRNA led to induction of p21/Cip1 and attenuation of G(1)-S transition in NSCLC cells. Using p21(+/+) and p21(-/-) HCT116 isogenic cell lines, we further showed that growth inhibition by PKC epsilon(KR) required the function of p21/Cip1. Collectively, these results reveal an important role for PKC epsilon signaling in lung cancer and suggest that one potential mechanism by which PKC epsilon exerts its oncogenic activity is through deregulation of the cell cycle via a p21/Cip1-dependent mechanism.

Differentiation of Human Embryonic Stem Cells into Immunostimulatory Dendritic Cells under Feeder-Free Culture Conditions

Purpose: The objective of this study was to develop a scalable and broadly applicable active immunotherapy approach against cancer, circumventing the limitations typically encountered with autologous vaccination strategies. We hypothesized that human embryonic stem cells (hESC) can serve as a virtually unlimited source for generating dendritic cells (DC) with potent antigen-presenting function. Here, we investigated the developmental processes and requirements for generating large numbers of mature, antigen-presenting DC from pluripotent hESC. Experimental Design: A feeder cell-free culture system was developed to differentiate hESC into mature DC sequentially through hematopoietic and myeloid precursor stages. Results: Using this method, we were able to yield large numbers of mature immunostimulatory DC from hESC to enable clinical investigation. Upon activation, the hESC-derived DC secreted interleukin-12p70, migrated in response to MIP-3β, and exhibited allostimulatory capacity. Most importantly, antigen-loaded, hESC-derived DC were capable of stimulating potent antigen-specific CD8+ T-cell responses in an HLA class I–matched semiallogeneic assay system. Moreover, HLA class II–mismatched hESC-derived DC induced a potent Th1-type cytokine response without expanding FOXP3+ regulatory T cells in vitro. Conclusions: These data suggest the development of a novel active immunotherapy platform to stimulate potent T-cell immunity in patients with intractable diseases, such as cancer or viral infection.

Cell-based selection provides novel molecular probes for cancer stem cells

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self‐renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell‐based Systematic Evolution of Ligands by EXponential enrichment (cell‐SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ∼90% and 5 bound to ∼15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E‐cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune‐compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.

Conditional Deletion of Jak2 Reveals an Essential Role in Hematopoiesis throughout Mouse Ontogeny: Implications for Jak2 Inhibition in Humans

Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at ∼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.

Abstract A5: Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate cancer

Purpose: SOX2 is a typical embryonic stem cell marker that is involved in not only tumorigenesis but also metastasis in several human malignancies including prostate cancer. Hypoxia has been demonstrated as a common feature of prostate cancer that is associated with disease progression and poor prognosis. Although SOX2 has been shown to be activated by oxygen depletion in other cancer types, its functional role under hypoxic microenvironment in prostate cancer setting and its association with hypoxia inducible factors (HIFs) have not been reported. In the present study, we evaluate the role of SOX2 in hypoxia-mediated cellular behaviors associated with metastasis, including invasion and sphere formation, in prostate cancer cells. Experimental Design: A tissue microarray containing samples from 80 prostate cancer patients was employed to examine the expression of HIF-1α and its correlation with SOX2 by immunohistochemical analysis. Expression of stem cell markers and HIFs was detected by Western blot. The role of SOX2 and HIF-1/2α in the regulation of cell invasion and sphere formation with various hypoxic exposures was investigated by short hairpin RNA (shRNA) - mediated knockdown in PC-3, DU145 and LNCaP cells. Results: HIF-1α expression was significantly elevated in prostate cancer compared to non-malignant prostate tissues, and in tumors HIF-1α was strongly correlated with SOX2. In all three prostate cancer cells, while HIF-1α and HIF-2α were mainly induced under short term (acute) and long term (chronic) hypoxic conditions, respectively, SOX2 was the only one among five stem cell markers that was stimulated by both hypoxic exposures. Genetic depletion of SOX2 attenuated capacities of tumor cells to invade under acute hypoxia and form spheroids under chronic hypoxia. In addition, knockdown of HIF-1α but not HIF-2α decreased acute hypoxia-mediated cell invasion, whereas only HIF-2α gene silencing resulted in reduced sphere formation capacity mediated by long term oxygen deprivation. Conclusions: SOX2 is suggested to be correlated with hypoxia in prostate cancer. SOX2 plays an important role in acute hypoxia-induced invasiveness and chronic hypoxia-induced stemness that is associated with HIF-1α and HIF-2α activation, respectively. These results indicate that SOX2 may be a key mediator for hypoxia-induced metastasis-associated functions hence may serve as a potential target for therapeutic interventions for advanced prostate cancer. Citation Format: Yao Dai, Kyung-Mi Bae, Johannes Vieweg, Dietmar W. Siemann. Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A5.

Abstract 485: Hypoxia increases the expression of stem-cell markers and promotes cell invasion in prostate cancer cells

Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). The existence of hypoxic microenvironment is a common occurrence in many solid tumors including prostate. Previous studies from our laboratories have shown that in human prostate cancer models, cancer stem cells expressing the embryonic stem cell markers SOX2 and OCT3/4 were highly invasive and, importantly, were capable of altering their E-cadherin expression during the process of invasion. In contrast, non-tumorigenic cells which minimally express SOX2 or OCT3/4 were found to be poorly invasive. Furthermore, targeted knockdown of SOX2 or OCT3/4 markedly suppressed the invasion of prostate cancer cells, suggesting an indispensible role of these two stem cell markers in invasiveness. In the present study, we examined the impact of oxygen deprivation on cell invasiveness and stemness in both stem cell enriched and non-stem cell populations in PC3 and DU145 prostate cancer cells. Moreover, the impact of Notch signaling, which has been shown as a critical regulator of prostate cancer development, progression and metastasis, was examined. When cells were exposed to 1% oxygen for 24 h, the invasive capacity of both the stem- and non-stem cell subpopulations increased. Interestingly the non-stem cell subpopulation showed the most striking increase (> 10-fold) in invasion under hypoxic conditions. The expression of several important stem cell factors, including OCT3/4, SOX2, Nanog and c-Myc, also was upregulated under hypoxia in both subpopulations. In addition, the expression of Notch1, Notch2 and their target genes Hey1, Hey2 and HeyL were increased in both subpopulations that had experienced oxygen deprivation (1% O 2 , 24 h). These findings suggest that although cancer stem cells per se exhibit significantly greater invasive capacity, aberrant microenvironmental conditions such as hypoxia may dramatically enhance the ability of non-stem cells to invade. Consequently, both the stem and non-stem cell subpopulations of tumors may be potential targets for the development of suitable therapeutic interventions designed to inhibit prostate cancer cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2011-485