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 þ CD24 À prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44 þ CD24 À cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44 þ CD24 À cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44 þ CD24 À 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 þ CD24 À 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 þ CD24 À cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44 þ CD24 À 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.

Aberrantly activated signal transducer and activator of transcription 3 (Stat3) is implicated in the development of various human cancers. Y705 phosphorylation is conventionally thought to be required for Stat3 signal-dependent activation and seems to play an essential role in some malignancies. Recently, it was shown that Stat3 is activated through novel and noncanonical mechanisms, including phosphorylation at S727. Here, we investigate S727 phosphorylation of Stat3 and its subsequent effects in prostate cancer development, independent of Y705 phosphorylation, using mutated Stat3 in the human prostate cancer cell line LNCaP. We show mutation of S727 to the phosphomimetic residue Glu, and inactivation of Y705 (Y705F/S727E) resulted in a remarkable growth advantage in low-serum, enhanced anchorage-independent growth in soft agar, and increased tumorigenicity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, possibly by direct activation of downstream proto-oncogenes c-myc, mcl-1, and survivin. Y705F/S727E mutant cells were more invasive than Y705F/ S727A (inactivation of Y705 and S727) mutant cells, and more Y705F/S727E mutant Stat3 was localized in the nuclei relative to Y705F/S727A mutant Stat3 at the steady state. Furthermore, the Y705F/S727E but not the Y705F/S727A mutant induced anchorage-independent growth of noncancerous prostate epithelial cells (RWPE-1). We further show that Stat3 is phosphorylated at S727 in 65% of malignant prostate tissues (n = 20) relative to 25% of normal prostate tissues (n = 4). Moreover, there is a positive correlation between phosphoS727-Stat3 expression and Gleason score in these prostate cancer tissues (P = 0.05). Our data suggest for the first time that S727 phosphorylation is sufficient to activate Stat3, thereby driving prostate tumorigenesis independent of Y705 phosphorylation.

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca 2؉ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca 2؉ store depletion. Apart from this mode of regulation, little is known about other receptordependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor-and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.In many types of cells, membrane receptors coupled to phospholipase C (PLC) 1 promote inositol 1,4,5-trisphosphate (IP 3 )-mediated release of Ca 2ϩ from endoplasmic reticulum and Ca 2ϩ entry across the plasma membrane through both capacitative or store-operated and non-capacitative calcium entry pathways (1-3). Although the molecular identity of capacitative and noncapacitative calcium entry channels, as well as the signaling mechanisms involved, remain uncertain, mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channel have been considered as potential candidates (4, 5), particularly members of the canonical TRP (TRPC) subfamily (designated TRPC1 through TRPC7 (6)). The human isoform of TRPC3, originally cloned by Zhu et al. (7), has been shown in many heterologous expression systems to behave as a receptoractivated channel that can be activated also by exogenous application of diacylglycerols (DAGs) independently of store depletion. In fact, DAG-induced activation of TRPC3 and its structural relatives TRPC6 and TRPC7 (8, 9) provides the likely mechanism of activation of these channels by phosphoinositide-specific PLClinked receptors, independently of IP 3 and store depletion. Apart from its regulation by DAG generated from PLC stimulation through either G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), little is known about additional signaling pathways downstream of receptor-stimulation involved in activation of TRPC3. Stimulation of either of these receptor pathways results in rapid tyrosine phosphorylation of cellular proteins (10, 11), and so in the present study we investigated whether tyrosine kinases might play a role in the signaling mechanism underlying receptor-and DAG-dependent activation of TRPC3. We found that inhibition of tyrosine kinase activity completely abrogated the ability of either GPCR st...

Cancer, second only to heart disease, is the leading cause of death in the US. Although progress has been made in the early detection of cancer and in improvements of cancer therapies, the ability to provide long-term survival has been limited. Increasing evidence suggests that a minute, biologically unique population of cancer stem cells (SCs) exists in most neoplasms and may be responsible for tumor initiation, progression, metastasis, and relapse. Characterization of cancer SCs has led to the identification of key cellular activities that may make cancer SCs vulnerable to therapeutic interventions that target drug-effluxing capabilities, stem cell pathways, anti-apoptotic mechanisms, and induction of differentiation. Phytochemicals, compounds made from fruits, vegetables, and grains, possess anti-cancer properties and represent a promising therapeutic approach for the prevention and treatment of many cancers. This review summarizes the evidence for the cancer SC hypothesis and discusses the potential mechanisms by which phytochemicals might target cancer SCs.

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Prostate cancer continues to represent a burgeoning medical problem in the United States. Recent studies suggest that gossypol, a bioactive phytochemical produced by cotton plants, is a promising agent against prostate cancer. The current studies were undertaken to examine the chemotherapeutic efficacy of gossypol on human prostate cancer cell lines and prostate tumor-initiating cells. Gossypol reduced the viability of three prostate cancer cell lines (LAPC4, PC3, and DU145) with an IC 50 between 3 and 5 μmol/L. Additionally, gossypol was effective at inhibiting prostate tumor-initiating cell-driven tumor growth in a nonobese diabetic/severe combined immunodeficient xenograft model. Our integrated molecular profiling approach encompassing proteomics, activated transcription factors, and genomics suggests that the decrease in viability was associated with increased DNA damage and the induction of apoptosis. Exposure of DU145 cells to gossypol (1-10 μmol/L) resulted in the activation of 13 proteins and 7 transcription factors, and the expression of 17 genes involved in the mitochondrial pathway of apoptosis. These studies show for the first time that gossypol treatment induces DNA damage and activates p53. Collectively, these data support the use of gossypol as a novel agent for prostate cancer. Mol Cancer Ther; 9(2); 461-70. ©2010 AACR.

Recent evidence suggests tumor-initating cells (TICs), also called cancer stem cells, are responsible for tumor initiation and progression; therefore, they represent an important cell population for development of future anti-cancer therapies. In this study, we show that the sesquiterpene lactone parthenolide (PTL) is cytotoxic to prostate TICs isolated from prostate cancer cell lines: DU145, PC3, VCAP and LAPC4, as well as primary prostate TICs. Furthermore, PTL inhibited TIC-driven tumor formation in mouse xenografts. Using an integrated molecular profiling approach encompassing proteomics, profiles of activated transcription factors and genomics we ascertained the effects of PTL on prostate cancer cells. In addition to the previously described effects of PTL, we determined that the non-receptor tyrosine kinase src, and many src signaling components, including: Csk, FAK, β1-arrestin, FGFR2, PKC, MEK/MAPK, CaMK, ELK-1 and ELK-1-dependent genes are novel targets of PTL action. Furthermore, PTL altered the binding of transcription factors important in prostate cancer including: C/EBP-α, fos related antigen-1 (FRA-1), HOXA-4, c-MYB, SNAIL, SP1, serum response factor (SRF), STAT3, X-box binding protein-1 (XBP1) and p53. In summary, we show PTL is cytotoxic to prostate TICs and describe the molecular events of PTL-mediated cytotoxicity. Therefore, PTL represents a promising therapeutic for prostate cancer treatment.

Tumor immunology fundamentals suggest immunological surveillance has the ability to recognize malignant cells and kill them before a tumor develops. However, cancer cells employ evasion mechanisms whereby the immune system may be actively suppressed or even tolerized to the tumor. Recently cancer stem cells were linked to tumor initiation and formation. However, no reports have addressed whether these cells participate in a tumor's ability to evade immune surveillance. Recently the glycoprotein CD200, expressed within the innate immune system and other tissues and cells, was shown to be involved in tolerance. Here we describe CD200 co-expression with stem cell markers found on prostate, breast, brain, and colon cancers. This is the first report describing an immunomodulatory molecule on epithelial cancer stem cells. This important finding suggests a mechanism by which a tumor might evades immune system detection.