Gossypol induces apoptosis by activating p53 in prostate cancer cells and prostate tumor-initiating cells.

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 micromol/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 micromol/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.

Effects of the sesquiterpene lactone parthenolide on prostate tumor-initiating cells: An integrated molecular profiling approach.

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, beta1-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-alpha, 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.

Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727 promotes prostate tumorigenesis independent of tyrosine 705 phosphorylation.

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.

Targeting cancer stem cells with phytochemicals.

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.

Cancer stem cells, CD200 and immunoevasion.

The limited success seen in cancer immunotherapy signifies that an alternative approach is required. Advances in cancer biology have identified a biologically unique subpopulation of cells, termed cancer stem cells (CSC), that survive after conventional therapy. CSCs are the putative cancer-initiating cells responsible for tumor initiation, progression and metastasis. CSCs might be able to evade the immune system by generating a tolerogenic response facilitated by the immunosuppressive factor CD200. This article reviews the biological importance of CSCs and the potentially important role of CD200 in tumor immunology. Moreover, we discuss the prospective role CD200 plays in the ability of a CSC to escape the immune system. Future immunotherapy must consider targeting CSCs to achieve curative responses.

Co-expression of the toleragenic glycoprotein, CD200, with markers for cancer stem cells.

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.

TGFBR3 loss and consequences in prostate cancer.

BACKGROUND: Resistance to transforming growth factor-beta (TGF-beta) is important in tumorigenesis. TGF-beta resistance mechanisms in prostate cancer are not well understood. METHODS: We have conducted a systematic analysis of TGF-beta pathway components with a meta-analysis of seven microarray studies using Oncomine and evaluated the results of TGFBR3 expression in prostate cell lines. Furthermore, we knocked down TGFBR3 in prostate epithelial cells and analyzed the consequences of TGFBR3 knockdown. RESULTS: We found that TGFBR3 is the TGF-beta component most commonly downregulated among localized human prostate cancer studies. TGFBR3 knockdown led to focus formation and enhanced expression of CD133, a marker found on prostate cancer stem cells. DNA microarray analysis of TGFBR3 knockdown cells identified 101 genes regulated by TGFBR3. Seven of these genes show a corresponding decrease in clinical prostate cancer specimens, which include genes involved in prostate mass and vasculature. CONCLUSIONS: TGFBR3 downregulation is an important step in prostate tumorigenesis.

Stem cells in prostate cancer: resolving the castrate-resistant conundrum and implications for hormonal therapy.

Androgen deprivation therapy (ADT) is initial systemic therapy for advanced prostate cancer and is used as an adjuvant to local therapy for high-risk disease, but responses in advanced disease are transient. Prostate cancer stem cells are a small fraction of tumor cells that give rise to malignant cells. Initial or acquired stem cell resistance to castration must therefore underlie castrate-resistant prostate cancer. We sought to review the evidence on cancer stem cells and androgen deprivation therapy to determine if prostate cancer stem cell resistance occurs from the outset, or if it is an acquired resistance. Prostate cancer stem cells do not express androgen receptor (AR) and hence should not be directly responsive to androgen deprivation therapy. However, castrate-resistant tumors that are derived from stem cells, have molecular changes such as amplification of the androgen receptor gene, or other genetic changes resulting in gain-of-function changes in AR, implying an acquired resistance to androgen deprivation. The origins of castrate-resistant tumors, with mechanisms such as androgen receptor gene amplification from androgen receptor negative prostate cancer stem cells, is an apparent conundrum. Insight into how this occurs may lead to new treatments that overcome or delay castrate-resistance. Herein, we review the evidence on cancer stem cells, the benefits of ADT, the biological basis of response to ADT, and mechanisms of castrate-resistance. We also explore the apparent conundrum of why AR-negative prostate cancer stem cells can give rise to castrate-resistant prostate cancer. We propose possible explanations that may resolve this conundrum and discuss implications for hormonal therapy.

Role of Src in C3 transient receptor potential channel function and evidence for a heterogeneous makeup of receptor- and store-operated Ca2+ entry channels.

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors and activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays we now report that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (i) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 had no effect; (ii) TRPC6 and TRPC7 were activated in src-, yes-, and fyn-deficient cells; and (iii) src, but not yes or fyn, rescued TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src was found to interact with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous.

Molecular cloning of TRPC3a, an N-terminally extended, store-operated variant of the human C3 transient receptor potential channel.

AK032317 is the GenBank accession no. of a full-length RIKEN mouse cDNA. It encodes a putative variant of the C3-type TRPC (transient receptor potential channel) that differs from the previously cloned murine TRPC3 cDNA in that it has a 5' extension stemming from inclusion of an additional exon (exon 0). The extended cDNA adds 62 aa to the sequence of the murine TRPC3. Here, we report the cloning of a cDNA encoding the human homologue of this extended TRPC3 having a highly homologous 73-aa N-terminal extension, referred to as hTRPC3a. A query of the GenBank genomic database predicts the existence of a similar gene product also in rats. Transient expression of the longer TRPC3a in human embryonic kidney (HEK) cells showed that it mediates Ca2+ entry in response to stimulation of the Gq-phospholipase C beta pathway, which is similar to that mediated by the shorter hTRPC3. However, after isolation of HEK cells expressing hTRPC3 in stable form, TRPC3a gave rise to Ca2+-entry channels that are not only activated by the Gq-phospholipase C beta pathway (receptor-activated Ca entry) but also by thapsigargin triggered store depletion. In conjunction with findings from our and other laboratories that TRPC1, TRPC2, TRPC4, TRPC5, and TRPC7, can each mediate store-depletion-activated Ca2+ entry in mammalian cells, our findings with hTRC3a support our previous proposal that TRPCs form capacitative Ca-entry channels.

Obligatory role of Src kinase in the signaling mechanism for TRPC3 cation channels.

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ 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 Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent 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.