Yelena Karpova

Epinephrine Protects Cancer Cells from Apoptosis via Activation of cAMP-dependent Protein Kinase and BAD Phosphorylation

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with β2-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.

Behavioral stress accelerates prostate cancer development in mice

Prostate cancer patients have increased levels of stress and anxiety. Conversely, men who take beta blockers, which interfere with signaling from the stress hormones adrenaline and noradrenaline, have a lower incidence of prostate cancer; however, the mechanisms underlying stress-prostate cancer interactions are unknown. Here, we report that stress promotes prostate carcinogenesis in mice in an adrenaline-dependent manner. Behavioral stress inhibited apoptosis and delayed prostate tumor involution both in phosphatase and tensin homolog-deficient (PTEN-deficient) prostate cancer xenografts treated with PI3K inhibitor and in prostate tumors of mice with prostate-restricted expression of c-MYC (Hi-Myc mice) subjected to androgen ablation therapy with bicalutamide. Additionally, stress accelerated prostate cancer development in Hi-Myc mice. The effects of stress were prevented by treatment with the selective β2-adrenergic receptor (ADRB2) antagonist ICI118,551 or by inducible expression of PKA inhibitor (PKI) or of BCL2-associated death promoter (BAD) with a mutated PKA phosphorylation site (BADS112A) in xenograft tumors. Effects of stress were also blocked in Hi-Myc mice expressing phosphorylation-deficient BAD (BAD3SA). These results demonstrate interactions between prostate tumors and the psychosocial environment mediated by activation of an adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway. Our findings could be used to identify prostate cancer patients who could benefit from stress reduction or from pharmacological inhibition of stress-induced signaling.

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.

Diverse Antiapoptotic Signaling Pathways Activated by Vasoactive Intestinal Polypeptide, Epidermal Growth Factor, and Phosphatidylinositol 3-Kinase in Prostate Cancer Cells Converge on BAD

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.

Epidermal Growth Factor Protects Prostate Cancer Cells from Apoptosis by Inducing BAD Phosphorylation via Redundant Signaling Pathways

Protection from apoptosis by receptor tyrosine kinases, resistant to the inhibition of phosphatidylinositol 3 ′-kinase/Akt and Ras/MEK pathways, has been reported in several cell types, including fibroblasts and epithelial prostate cancer cells; however, mechanisms of this effect were not clear. Here we report that in prostate cancer cells, epidermal growth factor activates two antiapoptotic signaling pathways that impinge on the proapoptotic protein BAD. One signaling cascade operates via the Ras/MEK module and induces BAD phosphorylation on Ser112. Another pathway predominantly relies on Rac/PAK1 signaling that leads to BAD phosphorylation on Ser136. Each of these two pathways is sufficient to protect cells from apoptosis, and therefore both have to be inhibited simultaneously to block epidermal growth factor-dependent survival. Redundancy of antiapoptotic signaling pathways should be considered when therapies targeting antiapoptotic mechanisms are designed.

BAD Dephosphorylation and Decreased Expression of MCL-1 Induce Rapid Apoptosis in Prostate Cancer Cells

PTEN loss and constitutive activation of the PI3K signaling pathway have been associated with advanced androgen-independent prostate cancer. PTEN-deficient prostate cancer C42Luc cells survive in serum-free media and show relative resistance to apoptosis even in the presence of the PI3K inhibitor ZSTK474. Yet, when ZSTK474 is combined with the translation inhibitor cycloheximide, C42Luc cells undergo apoptosis within 6 hours. We identified dephosphorylation of BAD (Bcl2-associated death promoter) as a main apoptosis-regulatory molecule downstream from PI3K, and loss of MCL-1 (Myeloid cell leukemia -1) as a major target of cycloheximide. The combination of MCL-1 knockdown and expression of phosphorylation-deficient mutant BAD2SA is sufficient to trigger rapid apoptosis in prostate cancer cells. These results establish the mechanism for the synergistic induction of apoptosis by the combination of a PI3K inhibitor and of a protein synthesis inhibitor in PTEN-deficient prostate cancer cells.

Surgical Stress Delays Prostate Involution in Mice

Androgens control growth of prostate epithelial cells and androgen deprivation induces apoptosis, leading to prostate involution. We investigated the effects of surgical stress on prostate involution induced by androgen ablation and determined the underlying mechanisms. Androgen ablation in mice was induced by surgical castration and administration of the anti-androgenic drugs bicalutamide and MDV3100. Surgical stress was induced by sham castration under isoflurane anesthesia. Surgical stress delayed apoptosis and prostate involution induced by anti-androgenic drugs. These effects of stress were prevented by administering the selective beta2-adrenoreceptor antagonist ICI118,551 and were also blocked in BAD3SA/WT mice expressing phosphorylation-deficient mutant BAD3SA. These results indicate that apoptosis and prostate involution in response to androgen ablation therapy could be delayed by surgical stress via the beta2-adrenoreceptor/BAD signaling pathway. Thus, surgery could interfere with androgen ablation therapy, whereas administration of beta2-adrenoreceptor antagonists may enhance its efficacy.

Synthesis and PI3 Kinase Inhibition Activity of Some Novel Trisubstituted Morpholinopyrimidines

A number of new substituted morpholinopyrimidines were prepared utilizing sequential nucleophilic aromatic substitution and cross-coupling reactions. One of the disubstituted pyrimidines was converted into two trisubstituted compounds which were screened as PI3K inhibitors relative to the well-characterized PI3K inhibitor ZSTK474, and were found to be 1.5–3-times more potent. A leucine linker was attached to the most active inhibitor since it would remain on any peptide-containing prodrug after cleavage by prostate-specific antigen, and it did not prevent inhibition of AKT phosphorylation and hence the inhibition of PI3K by the modified inhibitor.

Abstract 10: Effect of behavioral stress on therapeutic sensitivity of prostate cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Behavioral stress has been long implicated in cancer pathogenesis, yet mechanistic aspects of stress/tumor interactions are understudied. Several publications described effects of stress hormone epinephrine on tumor stroma components: vasculature and immune cells; however it was not clear whether epinephrine could influence cancer cells directly and if increase of epinephrine induced by behavioral stress could activate anti-apoptotic signaling in tumors and diminish efficacy of anti-cancer therapies. Here, we studied whether subjecting mice to behavioral stress increases resistance of PTEN-deficient prostate cancer to treatments with PI3K inhibitors. We examined effects of behavioral stress on C4-2Luc prostate cancer xenografts that express firefly luciferase and could be non-invasively monitored by luminescent imaging. In these tumor xenografts PI3K pathway is constitutively active due to the loss of PTEN expression. Injection of PI3K inhibitors into C4-2Luc xenografts induced apoptosis and substantially reduced luminescence. Subjecting mice to immobilization stress for 1 hour increased plasma epinephrine levels up to 10-15 nM, inhibited apoptosis and prevented reduction of luminescence in C4-2Luc xenografts treated with PI3K inhibitors. Injections of epinephrine had similar to stress effects. Conversely, the effect of either stress or epinephrine was completely blocked by beta2-adrenergic receptor (ADRB2) selective antagonist ICI118,551, implying that effects of stress/epinephrine are mediated via ADRB2. Elevated phosphorylation of BAD and CREB and inhibition of apoptosis was observed in xenograft tumors of mice subjected to immobilization stress. Effects of stress on BAD and CREB phosphorylation were completely blocked by ADRB2 selective antagonist ICI118,551. To test the role of protein kinase A (PKA) in anti-apoptotic signaling by stress/epinephrine in vivo we generated C4-2Luc cells that inducibly express chimera of PKA inhibitor peptide PKI and GFP (PKI-GFP). Induction of PKI-GFP expression in prostate cancer xenografts blocked stress/epinephrine-induced activation of PKA as judged by lack of CREB phosphorylation. It also prevented stress/epinephrine-induced activation of anti-apoptotic signals that phosphorylated BAD. Furthermore, C4-2Luc xenografts that inducibly express GFP-PKI were not protected from apoptosis by epinephrine or stress. In summary, we found that subjecting mice to behavioral stress resulted in increase of serum epinephrine level leading to activation of the ADRB2 and PKA pathway in prostate tumors. This ADRB2/PKA activation is necessary for in vivo BAD phosphorylation and protection of prostate tumors from apoptosis induced by PI3K inhibitors. Thus, elevated epinephrine levels could contribute to therapy resistance of prostate cancer. 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 10. doi:10.1158/1538-7445.AM2011-10

Abstract 183: Loss of Bad phosphorylation and Mcl-1 expression is necessary for rapid apoptosisinduced by combination of TGFα-PE and ZSTK474 in prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Advanced androgen-independent prostate cancer is notoriously resistant to conventional systemic therapies, and at the moment there is no effective protocol for hormone-refractory advanced prostate cancer affected patients. One of possible mechanisms of such resistance is activation of the anti-apoptotic signaling PI3K/Akt pathway that is constitutively active in the 60% of advanced prostate cancers. However, multiple inhibitors of PI3K are just well studied and some have just gone on to clinical trials, but unfortunately they showed limited efficacy against prostate cancer alone. Our recent experiments has shown remarkable synergy in inducing rapid and massive apoptosis when PI3K inhibitor ZSTK474 is used in combination with Pseudomonas aeruginosa exotoxin A fragment fused with Transforming Growth Factor Alpha (TGFα-PE38). Time lapse video microscopy of prostate cancer C4-2 cell line has shown the substantial cell death within 4-6 hours with combined administration, compared to limited cell death in single agents-treated cells. Analysis of PARP and cleaved-caspase 3 fragment by Western blotting confirmed that cell death occurs via apoptosis. Quantitation of caspase 3 and 7 activity with luminescent AFC-DEVD substrate confirmed synergy in apoptosis activation by combination of TGFα-PE38 and ZSTK474, while single agents even in higher concentration did not induce substantial apoptosis within 6 hours time period. Analysis of the mechanisms underlying this synergy has shown that PI3K inhibitor ZSTK474 triggers Bad dephosphorylation, while TGFα-PE38 reduces expression levels of Mcl-1. A slight increase of Bim protein was detected and related with ZSTK474 administration. No major variations were detected in the expression levels of pro- and anti-apoptotic proteins Bax, Bcl-2 and Bcl-XL. To address the role of Bad phosphorylation and Mcl-1 expression in apoptosis induction by PI3K inhibitor ZSTK474 and TGFα-PE38, we examined apoptosis in C4-2 cells that express phosphorylation-deficient mutant Bad (BAD2SA). Results indicated that the expression of BAD2SA sensitized C4-2 cells to apoptosis induced by TGFα-PE38. Experiments that address the role of Mcl-1 loss in apoptosis induced by TGFα-PE38 are ongoing. 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 183. doi:10.1158/1538-7445.AM2011-183