Prostate cancer cell-adipocyte interaction: leptin mediates androgen-independent prostate cancer cell proliferation through c-Jun NH2-terminal kinase

Prostate cancer is one of the leading causes of death among men in the United States, and acquisition of hormone resistance (androgen independence) by cancer cells is a fatal event during the natural history of prostate cancer. Obesity is another serious health problem and has been shown to be associated with prostate cancer. However, little is known about the molecular basis of this association. Here we show that factor(s) secreted from adipocytes stimulate prostate cancer cell proliferation. Leptin is one of the major adipose cytokines, and it controls body weight homeostasis through food intake and energy expenditure. We identify leptin as a novel growth factor in androgen-independent prostate cancer cell growth. Strikingly, leptin stimulates cell proliferation specifically in androgen-independent DU145 and PC-3 prostate cancer cells but not in androgen-dependent LNCaP-FGC cells, although both cell types express functional leptin receptor isoforms. c-Jun NH2-terminal kinase (JNK) has been shown recently to play a crucial role in obesity and insulin resistance. Intriguingly, leptin induces JNK activation in androgen-independent prostate cancer cells, and the pharmacological inhibition of JNK blocked the leptin stimulation of androgen-independent prostate cancer cell proliferation. This suggests that JNK activation is required for leptin-mediated, androgen-independent prostate cancer cell proliferation. Furthermore, other cytokines produced by adipocytes and critical for body weight homeostasis cooperate with leptin in androgen-independent prostate cancer cell proliferation: interleukin-6 and insulin-like growth factor I demonstrate additive and synergistic effects on the leptin stimulation of androgen-independent prostate cancer cell proliferation, respectively. Therefore, adipose cytokines, as well as JNK, are key mediators between obesity and hormone-resistant prostate cancer and could be therapeutic targets.     

Activation and cross-talk between Akt, NF-kappaB, and unfolded protein response signaling in 1-LN prostate cancer cells consequent to ligation of cell surface-associated GRP78

Binding of activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*) to cell surface-associated GRP78 on 1-LN human prostate cancer cells causes their proliferation. We have now examined the interplay between Akt activation, regulation of apoptosis, the unfolded protein response, and activation of NF-kappaB in alpha2M*-induced proliferation of 1-LN cells. Exposure of cells to alpha2M* (50 pM) induced phosphatidylinositol 3-kinase-dependent activation of Akt by phosphorylation at Thr-308 and Ser-473 with a concomitant 60-80% increase in Akt-associated kinase activity. ERK1/2 and p38 MAPK were also activated, but there was only a marginal effect on JNK activation. Treatment of 1-LN cells with alpha2M* down-regulated apoptosis and promoted NF-kappaB activation as shown by increases of Bcl-2, p-Bad(Ser-136), p-FOXO1(Ser-253), p-GSK3beta(Ser-9), XIAP, NF-kappaB, cyclin D1, GADD45beta, p-ASK1(Ser-83), and TRAF2 in a time of incubation-dependent manner. alpha2M* treatment of 1-LN cells, however, showed no increase in the activation of caspase -3, -9, or -12. Under these conditions, we observed increased unfolded protein response signaling as evidenced by elevated levels of GRP78, IRE1alpha, XBP-1, ATF4, ATF6, p-PERK, p-eIF2alpha, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of Akt(Thr-308), Akt(Ser-473), and IkappaB kinase alpha kinase. The effects of alpha2M* on the NF-kappaB activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, alpha2M* promotes cellular proliferation of 1-LN prostate cancer cells by activating MAPK and Akt-dependent signaling, down-regulating apoptotic signaling, and activating unfolded protein response signaling.     

Interplay between obesity and aging on myocardial geometry and function: Role of leptin-STAT3-stress signaling

BackgroundUncorrected obesity facilitates premature aging and cardiovascular anomalies. This study examined the interaction between obesity and aging on cardiac remodeling and contractile function. Methods: Cardiac echocardiographic geometry, function, morphology, intracellular Ca²⁺ handling, oxidative stress (DHE fluorescence), STAT3 and stress signaling were evaluated in young (3-mo) and old (12- and 18-mo) lean and leptin deficient ob/ob obese mice. Cardiomyocytes from young and old lean and ob/ob mice were treated with leptin (1 nM) for 4 h in vitro prior to assessment of mechanical and biochemical properties. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obese mice at young and old age were evaluated for comparison. Results: Our results displayed reduced survival in ob/ob mice. Obesity but less likely older age dampened echocardiographic, geometric, cardiomyocyte function and intracellular Ca²⁺ properties, elevated O₂^? and p^47phox NADPH oxidase levels with a more pronounced geometric change at older age. Immunoblot analysis revealed elevated p^47phox NADPH oxidase and dampened phosphorylation of STAT3, with a more pronounced response in old ob/ob mice, the effects were restored by leptin. Obesity and aging inhibited phosphorylation of Akt, eNOS, AMPK, and p38 while promoting phosphorylation of JNK and IκB. Leptin reconciled cardiomyocyte dysfunction, O₂^? yield, p^47phox upregulation, STAT3 dephosphorylation and stress signaling in ob/ob mice although its action on stress signaling cascades were lost at old age. High fat diet-induced and db/db obesity displayed aging-associated cardiomyocyte anomalies reminiscent of ob/ob model albeit lost leptin response.ConclusionsOur data suggest disparate age-associated obesity response in cardiac remodeling and contractile dysfunction due to phosphorylation of Akt, eNOS and stress signaling-related oxidative stress.     

Leptin Induces Cyclin D1 Expression and Proliferation of Human Nucleus Pulposus Cells via JAK/STAT,PI3K/Akt and MEK/ERK Pathways

Increasing evidence suggests that obesity and aberrant proliferation of nucleus pulposus (NP) cells are associated with intervertebral disc degeneration. Leptin, a hormone with increased circulating level in obesity, has been shown to stimulate cell proliferation in a tissue-dependent manner. Nevertheless, the effect of leptin on the proliferation of human NP cells has not yet been demonstrated. Here, we show that leptin induced the proliferation of primary cultured human NP cells, which expressed the leptin receptors OBRa and OBRb. Induction of NP cell proliferation was confirmed by CCK8 assay and immunocytochemistry and Real-time PCR for PCNA and Ki-67. Mechanistically, leptin induced the phosphorylation of STAT3, Akt and ERK1/2 accompanied by the upregulation of cyclin D1. Pharmacological inhibition of JAK/STAT3, PI3K/Akt or MEK/ERK signaling by AG490, Wortmannin or U0126, respectively, reduced leptin-induced cyclin D1 expression and NP cell proliferation. These experiments also revealed an intricate crosstalk among these signaling pathways in mediating the action of leptin. Taken together, we show that leptin induces human NP cell cyclin D1 expression and proliferation via activation of JAK/STAT3, PI3K/Akt or MEK/ERK signaling. Our findings may provide a novel molecular mechanism that explains the association between obesity and intervertebral disc degeneration.     

Leptin activates STAT and ERK2 pathways and induces gastric cancer cell proliferation

Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Here, we provide evidence that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolished leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduced leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduced cancer cell proliferation. Immunostaining of gastric cancer tissues displayed local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways and therefore blocking its action at the receptor level could be a rational therapeutic strategy.     

APPL1-Mediating Leptin Signaling Contributes to Proliferation and Migration of Cancer Cells

Leptin has been implicated in tumorigenesis and tumor progression, particularly in obese patients. As a multifunctional adaptor protein, APPL1 (containing pleckstrin homology domain, phosphotyrosine binding domain, and a leucine zipper motif 1) plays a critical role in regulating adiponectin and insulin signaling pathways. Currently, high APPL1 level has been suggested to be related to metastases and progression of some types of cancer. However, the intercourse between leptin signaling pathway and APPL1 remains poorly understood. Here, we show that the protein levels and phosphorylation statues of APPL1were highly expressed in tissues from human hepatocellular carcinoma and triple-positive breast cancer. Leptin stimulated APPL1 phosphorylation in a time-dependent manner in both human hepatocellular carcinoma HepG2 cell and breast cancer MCF-7 cell. Overexpression or suppression of APPL1 promoted or attenuated, respectively, leptin-induced phosphorylation of STAT3, ERK1/2, and Akt in the cancer cells, accompanied with enhanced or mitigated cell proliferation and migration. In addition, we identified that APPL1 directly bound to both leptin receptor and STAT3. This interaction was significantly enhanced by leptin stimulation. Our results suggested that APPL1 positively mediated leptin signaling and promoted leptin-induced proliferation and migration of cancer cells. This finding reveals a novel mechanism by which leptin promotes the motility and growth of cancer cells.     

Coordinate regulation of forskolin-induced cellular proliferation in macrophages by protein kinase A/cAMP-response element-binding protein (CREB) and Epac1-Rap1 signaling: effects of silencing CREB gene expression on Akt activation

In this study, we have examined the role of two cAMP downstream effectors protein kinase A (PKA) and Epac, in forskolin-induced macrophage proliferation. Treatment of macrophages with forskolin enhanced [(3)H]thymidine uptake and increased cell number, and both were profoundly reduced by prior treatment of cells with H-89, a specific PKA inhibitor. Incubation of macrophages with forskolin triggered the activation of Akt, predominantly by phosphorylation of Ser-473, as measured by Western blotting and assay of its kinase activity. Akt activation was significantly inhibited by LY294002 and wortmannin, specific inhibitors of phosphatidylinositol 3-kinase, but not by H-89. Incubation of macrophages with forskolin also increased Epac1 and Rap1.GTP. Immunoprecipitation of Epac1 in forskolin-stimulated cells co-immunoprecipitated Rap1, p-Akt(Thr-308), and p-Akt(Ser-473). Silencing of CREB gene expression by RNA interference prior to forskolin treatment not only decreased CREB protein and its phosphorylation at Ser-133, but also phosphorylation of Akt at Ser-473, and Thr-308. Concomitantly, this treatment inhibited [(3)H]thymidine uptake and reduced forskolin-induced proliferation of macrophages. Forskolin treatment also inhibited activation of the apoptotic mechanism while promoting up-regulation of the anti-apoptotic pathway. We conclude that forskolin mediates cellular proliferation via cAMP-dependent activation of both PKA and Epac.     

JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic

The molecular mechanisms by which arsenic (As³⁺) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As³⁺ on EZH2 phosphorylation and the signaling pathways important for As³⁺-induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As³⁺-induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As³⁺ can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As³⁺-activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As³⁺-induced EZH2 phosphorylation. Because As³⁺ is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As³⁺-induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As³⁺-induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As³⁺-induced carcinogenesis.