mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN^?/? LNCaP and androgen independent (AR?), PTEN^+/? DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (?) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.     

5alpha-androstane-3alpha,17beta-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is reduced from the potent androgen, 5alpha-dihydrotestosterone (5alpha-DHT), by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) in the prostate. 3alpha-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5alpha-DHT. However, 3alpha-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3alpha-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3alpha-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3alpha-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3alpha-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3alpha-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3alpha-HSD expression are significantly elevated in localized and advanced prostate cancer, 3alpha-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

5α-androstane-3α,17β-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5α-Androstane-3α,17β-diol (3α-diol) is reduced from the potent androgen, 5α-dihydrotestosterone (5α-DHT), by reductive 3α-hydroxysteroid dehydrogenases (3α-HSDs) in the prostate. 3α-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5α-DHT. However, 3α-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3α-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3α-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3α-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3α-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3α-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3α-HSD expression are significantly elevated in localized and advanced prostate cancer, 3α-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

STI571 reduces TRAIL-induced apoptosis in colon cancer cells: c-Abl activation by the death receptor leads to stress kinase-dependent cell death

Background

In an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.

Methods

Colon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.

Results

We found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.

Conclusions

All together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.     

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.     

Suppression versus induction of androgen receptor functions by the phosphatidylinositol 3-kinase/Akt pathway in prostate cancer LNCaP cells with different passage numbers

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway controls several important biological functions, such as cell growth regulation, apoptosis, and migration. However, the way in which PI3K/Akt controls androgen receptor (AR)-mediated prostate cancer cell growth remains unclear and controversial. Here, we demonstrate that the PI3K/Akt pathway regulates AR activity in a cell passage number-dependent manner. Specifically, PI3K/Akt pathway can suppress AR activity in androgen-dependent LNCaP cells with low passage numbers. In contrast, it can also enhance AR activity in LNCaP cells with high passage numbers. Furthermore, we also demonstrate that insulin-like growth factor-1 can activate the PI3K/Akt pathway that results in the phosphorylation of AR at Ser210 and Ser790. The consequence of these events may then change the stability of AR protein. Together, our results demonstrate that the PI3K/Akt pathway may have distinct mechanisms to modulate AR functions in various stages of prostate cancer cells and that a combined therapy of antiandrogens and anti-PI3K/Akt inhibitors may be worth considering as a future therapeutic approach to battle prostate cancer.     

Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.     

PC-1解除S6K对AKT信号通路的负反馈抑制

目的:通过建立过表达PC-1的前列腺癌LNCaP细胞系及敲低PC-1表达的C4-2细胞系,探究PC-1激活AKT信号通路的分子机制。方法:将PC-1基因及针对PC-1的siRNA序列,分别克隆至慢病毒表达载体pCDH-EF1-Myc-MCS-T2A-Puro及干扰载体pSIH1-H1-Puro,包装成慢病毒后分别感染前列腺癌LNCaP及C4-2细胞,通过Western印迹鉴定PC-1过表达及敲低效果,并检测PI3K/AKT/mTOR信号通路相关蛋白S6K、AKT的磷酸化水平。结果:PC-1过表达时,S6K磷酸化水平下降,而AKT的磷酸化水平上升。结论:PC-1可以通过抑制S6K激酶活性,解除其对AKT的负反馈抑制作用,从而激活AKT激酶的活性。     

Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis. Components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt). Also, mutations occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. These pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of elevated activated Akt levels to phosphorylate and inactivate Raf-1. We have investigated the genetic structures and functional roles of these two signaling pathways in the malignant transformation and drug resistance of hematopoietic, breast and prostate cancer cells. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell-lineage-specific effects. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines (FDC-P1 and TF-1), a trait associated with tumorigenesis. In contrast, expression of activated PI3K or Akt does not abrogate the cytokine dependence of these hematopoietic cell lines, but does have positive effects on cell survival. However, activated PI3K and Akt can synergize with activated Raf to abrogate the cytokine dependence of another hematopoietic cell line (FL5.12) which is not transformed by activated Raf expression by itself. Activated Raf and Akt also confer a drug-resistant phenotype to these cells. Raf is more associated with proliferation and the prevention of apoptosis while Akt is more associated with the long-term clonogenicity. In breast cancer cells, activated Raf conferred resistance to the chemotherapeutic drugs doxorubicin and paclitaxel. Raf induced the expression of the drug pump Mdr-1 (a.k.a., Pgp) and the Bcl-2 anti-apoptotic protein. Raf did not appear to induce drug resistance by altering p53/p21^Cip−1 expression, whose expression is often linked to regulation of cell cycle progression and drug resistance. Deregulation of the PI3K/PTEN/Akt pathway was associated with resistance to doxorubicin and 4-hydroxyl tamoxifen, a chemotherapeutic drug and estrogen receptor antagonist used in breast cancer therapy. In contrast to the drug-resistant breast cancer cells obtained after overexpression of activated Raf, cells expressing activated Akt displayed altered (decreased) levels of p53/p21^Cip−1. Deregulated expression of the central phosphatase in the PI3K/PTEN/Akt pathway led to breast cancer drug resistance. Introduction of mutated forms of PTEN, which lacked lipid phosphatase activity, increased the resistance of the MCF-7 cells to doxorubicin, suggesting that these lipid phosphatase deficient PTEN mutants acted as dominant negative mutants to suppress wild-type PTEN activity. Finally, the PI3K/PTEN/Akt pathway appears to be more prominently involved in prostate cancer drug resistance than the Raf/MEK/ERK pathway. Some advanced prostate cancer cells express elevated levels of activated Akt which may suppress Raf activation. Introduction of activated forms of Akt increased the drug resistance of advanced prostate cancer cells. In contrast, introduction of activated forms of Raf did not increase the drug resistance of the prostate cancer cells. In contrast to the results observed in hematopoietic cells, Raf may normally promote differentiation in prostate cells which is suppressed in advanced prostate cancer due to increased expression of activated Akt arising from PTEN mutation. Thus in advanced prostate cancer it may be advantageous to induce Raf expression to promote differentiation, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK-induced proliferation. These signaling and anti-apoptotic pathways can have different effects on growth, prevention of apoptosis and induction of drug resistance in cells of various lineages which may be due to the expression of lineage-specific factors.     

Signaling pathways that control the growth and survival of prostate tumor cells in the absence of androgens

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-alpha and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-kappa B, STAT3, PI3K, and beta-catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.     

Inhibition of Proliferation and Induction of Autophagy by Atorvastatin in PC3 Prostate Cancer Cells Correlate with Downregulation of Bcl2 and Upregulation of miR-182 and p21

The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in a dose- and time-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells.     

Combined AKT and MEK Pathway Blockade in Pre-Clinical Models of Enzalutamide-Resistant Prostate Cancer

Despite recent improvements in patient outcomes using newer androgen receptor (AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer (CRPC) continues to remain a clinical problem. Co-targeting alternate resistance pathways are of significant interest to treat CRPC and delay the onset of resistance. Both the AKT and MEK signaling pathways become activated as prostate cancer develops resistance to AR-targeted therapies. This pre-clinical study explores co-targeting these pathways in AR-positive prostate cancer models. Using various in vitro models of prostate cancer disease states including androgen dependent (LNCaP), CRPC (V16D and 22RV1) and ENZ-resistant prostate cancer (MR49C and MR49F), we evaluate the relevance of targeting both AKT and MEK pathways. Our data reveal that AKT inhibition induces apoptosis and inhibits cell growth in PTEN null cell lines independently of their sensitivity to hormone therapy; however, AKT inhibition had no effect on the PTEN positive 22RV1 cell line. Interestingly, we found that MEK inhibition had greater effect on 22RV1 cells compared to LNCaP, V16D or ENZ-resistant cells MR49C and MR49F cells. In vitro, combination AKT and MEK blockade had evidence of synergy observed in some cell lines and assays, but this was not consistent across all results. In vivo, the combination of AKT and MEK inhibition resulted in more consistent tumor growth inhibition of MR49F xenografts and longer disease specific survival compared to AKT inhibitor monotherapy. As in our in vitro study, 22RV1 xenografts were more resistant to AKT inhibition while they were more sensitive to MEK inhibition. Our results suggest that targeting AKT and MEK in combination may be a valuable strategy in prostate cancer when both pathways are activated and further support the importance of characterizing the dominant oncogenic pathway in each patient’s tumor in order to select optimal therapy.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Roles of the PI3K/Akt pathway and autophagy in TLR3 signaling-induced apoptosis and growth arrest of human prostate cancer cells

Toll-like receptors (TLRs) are widely expressed in immune cells and play a crucial role in many aspects of the immune response. Although some types of TLRs are also expressed in cancer cells, the effects and mechanisms of TLR signaling in cancer cells have not yet been fully elucidated. In the present study, we analyzed the effects of polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 ligand, on three TLR3-expressing human prostate cancer cell lines (LNCaP, PC3, and DU145). We then further characterized the underlying mechanisms, focusing on the poly(I:C)-sensitive LNCaP cell line. Poly(I:C) significantly reduced the viability of LNCaP cells TLR3 and endosome dependently. One mechanism for the antitumor effect was caspase-dependent apoptosis, and another mechanism was poly(I:C)-induced growth arrest. Cell survival and proliferation of LNCaP cells depended on the PI3K/Akt pathway, and PI3K/Akt inhibitors induced apoptosis and growth arrest similar to poly(I:C) treatment. Additionally, poly(I:C) treatment caused dephosphorylation of Akt in LNCaP cells, but transduction of the constitutively active form of Akt rendered LNCaP cells resistant to poly(I:C). Immunoblot analysis of proliferation- and apoptosis-related molecules in poly(I:C)-treated LNCaP cells revealed participation of cyclinD1, c-Myc, p53, and NOXA. Interestingly, poly(I:C) treatment of LNCaP cells was accompanied by autophagy, which was cytoprotective toward poly(I:C)-induced apoptosis. Together, these findings indicate that TLR3 signaling triggers apoptosis and growth arrest of LNCaP cells partially through inactivation of the PI3K/Akt pathway and that treatment-associated autophagy plays a cytoprotective role.