We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinasedefective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60 v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70 S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinasedead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.

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 β 2 -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 (BAD S112A ) in xenograft tumors. Effects of stress were also blocked in Hi-Myc mice expressing phosphorylation-deficient BAD (BAD 3SA ). 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.

Although a causal role of genetic alterations in human cancer is well established, it is still unclear whether dietary fat can modulate cancer risk in a predisposed population. Epidemiological studies suggest that diets rich in omega-3 polyunsaturated fatty acids reduce cancer incidence. To determine the influence of fatty acids on prostate cancer risk in animals with a defined genetic lesion, we used prostate-specific Pten-knockout mice, an immune-competent, orthotopic prostate cancer model, and diets with defined polyunsaturated fatty acid levels. We found that omega-3 fatty acids reduced prostate tumor growth, slowed histopathological progression, and increased survival, whereas omega-6 fatty acids had opposite effects. Introducing an omega-3 desaturase, which converts omega-6 to omega-3 fatty acids, into the Pten-knockout mice reduced tumor growth similarly to the omega-3 diet. Tumors from mice on the omega-3 diet had lower proportions of phosphorylated Bad and higher apoptotic indexes compared with those from mice on omega-6 diet. Knockdown of Bad eliminated omega-3-induced cell death, and introduction of exogenous Bad restored the sensitivity to omega-3 fatty acids. Our data suggest that modulation of prostate cancer development by polyunsaturated fatty acids is mediated in part through Bad-dependent apoptosis. This study highlights the importance of gene-diet interactions in prostate cancer.

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 ␤ 2 -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.Epinephrine levels are sharply increased in response to acute stress and can be continuously elevated during persistent stress and depression (1, 2). Sustained increases of epinephrine were implicated in pathogenesis of stress-related immunosuppression proposed as the primary mechanism by which stress and depression may increase tumor incidence and promote metastatic growth (2, 3). However, several reports have questioned whether immunosuppression alone is sufficient to explain stress-induced tumor growth, and some studies have found no correlation between stress and cancer (2, 4). Thus, more information about the mechanisms by which stress hormones affect tumors is necessary to resolve the controversy over the connection between stress and cancer. One potential mechanism may involve direct effects of epinephrine on cancer cells.Cancer cell lines of various origins, including prostate tumors, express ␤ 2 -adrenergic receptors (␤ 2 -ARs) 4 that bind epinephrine and norepinephrine (5-7). ␤ 2 -ARs belong to superfamily A of seven-transmembrane G protein-coupled receptors (GPCRs) (8). Epinephrine binding leads to activation of GTPase and dissociation of ␣ and ␤␥ subunits of heterotrimeric G proteins. Depending on the cell context, this may trigger multiple signaling pathways, including the Ras/extracellular signal-regulated kinase, NFB, and cAMP-dependent protein kinase (PKA) pathways, which regulate diverse cellular responses, such as proliferation, differentiation, secretion, or apoptosis (9).Since resistance to apoptosis has been implicated in cancer pathogenesis (10), we decided to analyze the effects of the ␤ 2 -AR agonist epinephrine on apoptosis in prostate cancer cells. In this paper, we demonstrate that epinephrine reduces sensitivity of prostate cancer cells to apoptosis via ␤ 2 -AR/PKA signaling that triggers BAD phosphorylation at S112. This antiapoptotic mechanism operates in the prostate cancer cell lines LNCaP and C4-2 and in the breast cancer cell line MDA-MB231. Our findings suggest that stress may contribute to cancer etiology and therapeutic resistance by decreasing sensitivity of cancer cells to apoptosis. EXPERIMENTAL PROCEDURESCell Lines and Transfection-LNCaP and C4-2 cells were a gift from Leland C...

Protein kinase B (PKB)/Akt is implicated in survival signaling in a wide variety of cells including fibroblasts and epithelial and neuronal cells. We and others have described a linear survival signaling cascade used by insulinlike growth factor I (IGF-I) that consists of the IGF-I receptor, phosphoinositide 3-kinase (PI3 kinase), Akt, and Bad. Activation of this pathway can be sufficient to protect cells from apoptosis. However, previous work had not determined whether this pathway is invariably necessary for protection from apoptosis or whether there are alternative survival signaling pathways. In this communication, we report the existence of two survival signaling pathways, one dependent on PI3 kinase and Akt and the other independent of these enzymes. We found that survival signaling initiated by IGF-I treatment of Rat-1 cells could be blocked by overexpression of a dominant negative kinase-deficient Akt (K179A) as well as by wortmannin. This demonstrates a survival signaling pathway dependent on PI3 kinase and Akt. However, when IGF-I receptors were overexpressed in a Rat-1 background (RIG cells), an alternative pathway became apparent, in which survival mediated by IGF-I was no longer sensitive to wortmannin or to overexpression of dominant negative Akt, even though Akt activation and Bad phosphorylation were still wortmannin sensitive. Experiments with inhibitors of RNA synthesis showed that transcriptional activation is dispensable for this alternative PI3 kinase/Akt-independent survival signaling. These findings demonstrate the existence of a new survival signaling pathway independent of PI3 kinase, Akt, and new transcription and which is evident in fibroblasts overexpressing the IGF-I receptor.The last several years have seen remarkable advances in understanding the machinery of apoptosis and the factors initiating the cascade of events leading to apoptotic cell death (7). In contrast, only recently has equivalent attention been paid to the ways that the probability of apoptosis is regulated in response to cellular physiology. Although it has been known for some time that cytokines and growth factors such as interleukin-2 (IL-2), IL-3, nerve growth factor, and insulinlike growth factor I (IGF-I) promote survival in various experimental cell systems, it was not clear which signaling pathways were used by these agents (3). One of the first reports on survival signaling connected activation of the mitogen-activated protein (MAP) kinase cascade with survival in PC-12 cells (44). Another signaling pathway requiring phosphoinositide 3-kinase (PI3 kinase) activity was associated with antiapoptotic signaling in neurons, fibroblasts, and hematopoietic cells (30,45,46). Subsequently, the serine/threonine kinase protein kinase B (PKB)/ Akt was identified as a downstream component of survival signaling through PI3 kinase (11,(17)(18)(19)24). Recently Bad, a proapoptotic member of the bcl-2 family, was found to be a substrate of Akt, identifying an intersection point of pro-and antiapoptotic regulatory cascades (8...

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.

Insulin and the insulin-like growth factor type I (IGF-I) 1 are peptide hormones that regulate distinct biological functions through interaction with their cognate receptors (Drop et al.,

Emerging evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies is one of the major limiting factors of cancer therapy efficacy. Identification of mechanisms responsible for survival and self-renewal of CSC will help design new therapeutic strategies that target and eliminate both differentiated cancer cells and CSC. Here we demonstrated the potential role of proapoptotic protein BAD in the biology of CSC in melanoma, prostate and breast cancers. We enriched CD44 þ /CD24 À cells (CSC) by tumorosphere formation and purified this population by FACS. Both spheres and CSC exhibited increased potential for proliferation, migration, invasion, sphere formation, anchorage-independent growth, as well as upregulation of several stem cell-associated markers. We showed that the phosphorylation of BAD is essential for the survival of CSC. Conversely, ectopic expression of a phosphorylation-deficient mutant BAD induced apoptosis in CSC. This effect was enhanced by treatment with a BH3-mimetic, ABT-737. Both pharmacological agents that inhibit survival kinases and growth factors that are involved in drug resistance delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. Furthermore, the frequency and self-renewal capacity of CSC was significantly reduced by knocking down the BAD expression. Consistent with our in vitro results, significant phosphorylation of BAD was found in CD44 þ CSC of 83% breast tumor specimens. In addition, we also identified a positive correlation between BAD expression and disease stage in prostate cancer, suggesting a role of BAD in tumor advancement. Our studies unveil the role of BAD in the survival and self-renewal of CSC and propose BAD not only as an attractive target for cancer therapy but also as a marker of tumor progression.