Suneetha B. Thomas

CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis.

Recent evidence supports the hypothesis that cancer stem cells are responsible for tumour initiation and formation. Using flow cytometry, we isolated a population of CD44+CD24− prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44+CD24− cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44+CD24− cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44+CD24− prostate stem-like cells as nonadherent spheres in serum-replacement media without substantially shifting gene expression. Addition of serum results in adherence to plastic and shifts gene expression patterns to resemble the differentiated parental cells. Thus, we propose that CD44+CD24− prostate cells are stem-like cells responsible for tumour initiation and we provide a genomic definition of these cells and the differentiated cells they give rise to. Furthermore, gene expression patterns of CD44+CD24− cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44+CD24− LNCaP prostate cells offer an attractive model system to both explore the biology important to the maintenance and differentiation of prostate cancer stem cells as well as to develop the therapeutics, as the gene expression pattern in these cells is consistent with poor survival in prostate cancer patients.

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44+ cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positve and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44+ CSC-like cells invade Matrigel through an EMT, while in contrast, CD44− cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44+CD24− prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.

BMI1 silencing enhances docetaxel activity and impairs antioxidant response in prostate cancer

The BMI1 oncogene promotes prostate cancer (PC) progression. High B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI1) expression predicts poor prognosis in PC patients. Recent evidence suggests that BMI1 may also play a role in docetaxel chemoresistance. However, mechanisms and clinical significance of BMI1‐related chemoresistance have not been investigated. For this purpose, BMI1 was silenced in 2 PC cell lines (LNCaP and DU 145). Cell proliferation and apoptosis after docetaxel treatment were measured. Guanine oxidation was assessed by in‐cell western. Global gene expression analysis was performed on BMI1 silenced cells. Oncomine database was used to compare in vitro data with gene expression in PC samples. BMI1 silencing had no effect on cell proliferation but significantly enhanced docetaxel‐induced antitumor activity. Gene expression analysis demonstrated that BMI1 silencing downregulates a set of antioxidant genes. Docetaxel treatment increased guanine oxidation, whereas the antioxidant N‐acetyl cysteine rescued docetaxel‐induced cell death. Examination of clinical datasets revealed a positive correlation of BMI1 and antioxidant gene expression. BMI1‐controlled antioxidant genes were predictive of poor prognosis in PC patients. In conclusion, BMI1 enhances antioxidant response, thereby allowing PC survival after docetaxel‐based chemotherapy. BMI1‐controlled antioxidant genes are overexpressed in aggressive PC and should be tested as predictors of chemotherapy failure.

Identification of Vitronectin as an Extrinsic Inducer of Cancer Stem Cell Differentiation and Tumor Formation

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self‐renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin αVβ3‐dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of β‐catenin nuclear localization. Blocking integrin αVβ3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways. STEM CELLS 2010;28:390–398

Molecular consequences of SOD2 expression in epigenetically silenced pancreatic carcinoma cell lines

Manganese superoxide dismutase (SOD2) is an enzyme that catalyses the dismutation of superoxide in the mitochondria, leading to reduced levels of reactive oxygen species. Reduced expression levels of SOD2 have been shown to result in increased DNA damage and sod2 heterozygous mice have increased incidences of cancer. It has also been shown that SOD2 expression is lost in pancreatic cell lines, with reintroduction of SOD2 resulting in decreased rate of proliferation. The mechanism of decreased SOD2 expression in pancreatic carcinoma has not been previously determined. We demonstrate, through sodium bisulphite sequencing, that the sod2 locus is methylated in some pancreatic cell lines leading to a corresponding decrease in SOD2 expression. Methylation can be reversed by treatment with zebularine, a methyltransferase inhibitor, resulting in restored SOD2 expression. Furthermore, we demonstrate that sensitivity of pancreatic carcinoma cell lines to 2-methoxyestradiol correlates with SOD2 expression and SOD2 modulation can alter the sensitivity of these cells. Using both genomics and proteomics, we also identify molecular consequences of SOD2 expression in MIA-PaCa2 cells, including dephosphorylation of VEGFR2 and the identification of both SOD2-regulated genes and transcription factors with altered binding activity in response to SOD2 expression.

Epigenetic silencing of manganese superoxide dismutase (SOD-2) in KAS 6/1 human multiple myeloma cells increases cell proliferation.

The generation of reactive oxygen species (ROS) by mitochondrial electron transport chain (ETC) and oxidative phosphorylation activity, has been linked to modifications of multiple molecular processes, including lipid peroxidation, signaling pathway and transcription factor modulation, and oxidative damage to DNA. Oxidative damage by endogenous ROS has been associated with the etiology of various pathological states. There are numerous reports that levels of manganese superoxide dismutase enzyme (MnSOD), an antioxidant enzyme responsible for the attenuation of ROS, are lowered in cancer cells, but the reasons for this reduction are poorly defined. Epigenetic silencing of genes involved in tumor suppression and DNA repair is known to occur in a variety of malignant cell types. Here we report that in the human multiple myeloma cell line KAS 6/1, the SOD-2 gene, encoding manganese superoxide dismutase, is epigenetically silenced as a result of promoter hypermethylation. The DNA methyltransferase inhibitor Zebularine reverses SOD-2 promoter methylation, increasing gene expression and enzyme levels. Infection of KAS 6/1 cells with a recombinant adenovirus carrying the MnSOD cDNA reduced the cell proliferation rate by approximately one-half, confirming the detrimental effects of epigenetic silencing of SOD-2 expression.

Effects of Manganese Superoxide Dismutase Silencing on Androgen Receptor Function and Gene Regulation: Implications for Castration-Resistant Prostate Cancer

Purpose: Advanced prostate cancer is first treated with androgen deprivation therapy. However, tumors become resistant to and grow despite castrate levels of testosterone. Growth and proliferation of CRPC is mediated by gain-of-function changes in the AR and AR reactivation. Expression of manganese superoxide dismutase (SOD2), which regulates cellular ROS, is markedly down-regulated in CRPC when compared with hormone-responsive tumors. Experimental Design: Here, we knocked down SOD2 expression in AR-expressing LNCaP prostate cancer cells and determined gene expression changes, transcription factor binding, and AR transcription activity in SOD2 knockdown cells. Results: SOD2 knockdown results in an increase in ROS. Gene expression changes induced by SOD2 knockdown results in the up-regulation of genes that are also androgen responsive and 46% of genes up-regulated 2-fold by the androgen ligand R1881 are also up-regulated to the same extent with SOD2 knockdown. The induction of many of these genes with SOD2 knockdown, such as VEGFA and FKBP5, is reversible with the antioxidant N-acetylcysteine, suggesting that this mechanism is directly linked to ROS. Furthermore, an array for transcription factor DNA-binding activity shows that SOD2 knockdown induces DNA binding by several transcription factors, including AR. SOD2 knockdown-induced AR activation was confirmed by electrophoretic mobility shift assay and luciferase activity, and both were readily reversible with N-acetylcysteine. Conclusions: These findings show that down-regulation of SOD2 induces AR activity in a ROS-dependent manner, and suggest that there may be a role for antioxidant therapy in CRPC.

Reversal of p53 epigenetic silencing in multiple myeloma permits apoptosis by a p53 activator.

Inactivation of the p53 pathway is a common feature of neoplasia. Dysregulation of the p53 pathway has been shown to involve mutations of p53, increased expression of the p53 inhibitor HDM-2, or epigenetic silencing of the p53 promoter. In multiple myeloma, a neoplasia of terminally differentiated B cells, p53 mutations and deletions are relatively rare and occur in late stage disease. Here, we show that the p53 promoter is hypermethylated in several multiple myeloma cell lines in comparison to normal plasma cells. Two cell lines containing mutant p53, Lp-1 and OPM-2, show a methylation pattern that suggests that they contain one methylated and one unmethylated mutant allele. Two other cell lines, KMS-11 and OPM-2, show hypermethylation of p53 with a lack of expression. In all cell lines tested, treatment with a demethylating agents results in higher expression of p53. Furthermore, following increased expression of p53, treatment of the myeloma cell lines with a p53 activating peptide induces apoptosis. Therefore, combinatorial treatment with a demethylating agent followed by delivery of a p53 activating peptide may be an effective therapeutic strategy against multiple myeloma.

DNA hypermethylation and partial gene silencing of human thymine- DNA glycosylase in multiple myeloma cell lines.

Multiple myeloma (MM) has prominent features of karyotypic instability at the earliest stage, leading to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways. A possible mechanism of dysregulation of the DNA repair pathway is epigenetic gene silencing. Therefore, we sought to determine the methylation status of enzymes involved in the base excision repair pathway in multiple myeloma cell lines. Here, we report the aberrant DNA methylation of TDG, one of the enzymes involved in base excision repair of damaged DNA, in several multiple myeloma cell lines but not in normal human plasma cells. DNA hypermethylation of TDG in the MM cell lines leads to lower gene expression levels that results in less efficient DNA repair activity in response to hydrogen peroxide-induced DNA damage. Expression of exogenous TDG can functionally compensate for lower repair activities of damaged DNA in the KAS-6/1 myeloma cell line, which has extensive DNA hypermethylation of the TDG promoter. Hypermethylation of DNA damage repair genes in MM cell lines may provide an explanation for the frequent genomic instability, as well as point mutations, that are encountered in MM.

The differentiation of pancreatic tumor-initiating cells by vitronectin can be blocked by cilengitide.

Objective Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as most of the patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells (TICs) constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis, and chemo/radioresistance. Within pancreatic cancer, TICs have been identified based on the expression of specific cell surface markers. Methods We use a sphere formation assay to enrich putative TICs and use human serum as a driver of differentiation. We demonstrate by using specific blocking reagents that we can inhibit the differentiation process and maintain TIC-associated markers and genes. Results We can induce differentiation of pancreatospheres with the addition of human serum, and we identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate–specific peptide, which is Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors. Conclusions Overall, our studies further the definition of pancreatic TICs and provide further insight into both the maintenance and differentiation of this lethal population.