Extracellular calcium and calcium sensing receptor function in human colon carcinomas: promotion of E-cadherin expression and suppression of beta-catenin/TCF activation

Ca(2+) has chemopreventive activity against colon cancer, albeit its mechanisms of action are not understood. In this study, we showed that four different human colon carcinoma cell lines (FET, SW480, MOSER, and CBS) expressed the human parathyroid calcium sensing receptor (CaSR) and that a function of extracellular Ca(2+) and the CaSR in these cells was the promotion of E-cadherin expression and suppression of beta-catenin/T cell factor activation. We also found that human colonic crypt epithelial cells expressed the CaSR, and histologically differentiated carcinomas (i.e., where three-dimensional, crypt-like structures were present) expressed less receptor by comparison, whereas an almost complete loss of CaSR expression was observed in undifferentiated tumors. These results suggest that extracellular Ca(2+) and the CaSR may function to regulate the differentiation of colonic epithelial cells and that disruption of this ligand receptor system may contribute to abnormal differentiation and malignant progression. In addition, the promotion of E-cadherin and suppression of beta-catenin/T cell factor may be an important mechanism underlying the chemopreventive action of Ca(2+) in colon cancer.     

Modulation of cyclin D1 expression in human tumoral parathyroid cells: effects of growth factors and calcium sensing receptor activation

The study investigated cyclin D1 regulation by growth factors and calcium sensing receptor (CaSR) in human tumoral parathyroid cells. Basic fibroblast and epidermal growth factors increased cyclin D1 and phosphorylated extracellular signal-regulated kinases (pERK1/2) levels that were both efficiently inhibited by CaSR agonists. By contrast, in growth factors-free medium cyclin D1 levels were either unaffected or stimulated by CaSR activation independently from ERK1/2 pathway. Transforming growth factor beta (TGFbeta) reduced cyclin D1 levels in the majority of tumors, this effect being not influenced by CaSR activation and menin expression levels. In conclusion, in parathyroid tumors cyclin D1 expression was modulated by growth factors and CaSR activation. These data further support the oncogenic role of cyclin D1, which resulted to be target for stimulation by bFGF and EGF and inhibition by CaSR and TGFbeta signalling in the parathyroid.     

Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: promotion of cytotoxic response to mitomycin C and fluorouracil

Ca(2+) and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca(2+) (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs.     

Nifedipine synergizes with calcium in activating the calcium sensing receptor, suppressing the expression of thymidylate synthase and survivin and promoting sensitivity to fluorouracil in human colon carcinoma cells

We have previously reported that activation of the G protein coupled calcium-sensing receptor (CaSR) by extracellular Ca(2+) down-modulates the expression of thymidylate synthase (TS) and survivin and promotes sensitivity to fluorouracil in human colon carcinoma cells. Here, we report for the first time that the cardiac drug nifedipine acted synergistically with Ca(2+) in CaSR activation and in the induction of intracellular Ca(2+). Nifedipine in combination with Ca(2+) significantly down-modulated the expression of TS and survivin and promoted sensitivity to 5-FU above and beyond the level achievable with Ca(2+) alone. Nifedipine by itself, however, had no effect on the suppression of TS or survivin or sensitivity to 5-FU. The action of Ca(2+) or in combination with nifedipine was entirely CaSR dependent as the aforementioned effects did not occur in CaSR knocked down cells. siRNAs targeting TS or survivin or both could mimic the effect of CaSR activation in promoting sensitivity to 5-FU. We conclude that nifedipine acts in synergy with Ca(2+) in activating CaSR and in promoting sensitivity to 5-FU by down modulating the expression of TS and survivin. G-protein coupled CaSR has the potential of serving as a target for improving therapeutic outcome in colon cancer.     

Inhibition of phorbol ester-induced phenotypic differentiation of HL-60 cells by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor

To identify the possible role of calcium ions in cell differentiation, we studied the extracellular Ca2+ requirement and the effect of Ca2+/phospholipid-dependent protein kinase (protein kinase C) inhibitor on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. HL-60 cells grew equally well in 0.1 and 1.0 mM Ca2+ media. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,25-dihydroxyvitamin D3, and all-trans-beta-retinoic acid inhibited the cell growth and induced mature macrophage and granulocyte phenotypes in 1.0 mM Ca2+ medium. 1,25-Dihydroxyvitamin D3 and all-trans-beta-retinoic acid induced HL-60 differentiation to the same degree in 0.1 mM Ca2+ and 1.0 mM Ca2+ media. However, TPA failed to induce HL-60 differentiation or to inhibit proliferation in a 0.1 mM Ca2+ medium. The decrease of extracellular Ca2+ from 1.0 to 0.1 mM caused a significant drop in the intracellular Ca2+ level in undifferentiated and TPA-treated HL-60 cells, although no rapid change in cytosolic Ca2+ was detected in response to TPA addition. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, inhibited proliferation of HL-60 cells in a dose-dependent manner. In contrast, H-7 selectively restored the proliferation of TPA-treated HL-60 cells and inhibited TPA-induced phenotypic differentiation. However, the same concentrations of 1-(5-isoquinolinylsulfonyl)-2,3-dimethylpiperazin and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, analogues of H-7 that inhibit protein kinase C more weakly, had no effect on the proliferation or differentiation induction. H-7 also suppressed 1,25-dihydroxyvitamin D3- and all-trans-beta-retinoic acid-induced phenotypic changes of HL-60 cells but did not eliminate the growth inhibition by these inducers. These results demonstrate the Ca2+ requirement and the protein kinase C involvement in phorbol ester-induced phenotypic differentiation of HL-60 cells.     

Role of basal calcium in the EGF activation of MAP kinases

The role of intracellular Ca2+ pools in the regulation of growth factor signal transduction pathways and mitogenesis is not well understood. We have examined the roles of basal and transiently mobilized Ca2+ in the regulation of MAP kinases by EGF. To assess the influence of Ca2+ transients we utilized Plcg1-/- and Plcg1+/+ mouse embryonic fibroblasts, while BAPTA/AM was employed to chelate total intracellular Ca2+ in the same cell lines. The MAP kinases erk-1, erk-2 and erk-5 exhibited similar patterns of activation in wild-type and Plcg1-/- cells treated with EGF. However, pretreatment with BAPTA/AM significantly increased and prolonged erk-1 and erk-2 activation in both cell types. In contrast, BAPTA/AM prevented the EGF activation of erk-5 in wild-type and Plcg1-/- cells. These data indicate that basal Ca2+, but not growth factor provoked Ca2+ transients, has a significant influence on the activation of these MAP kinases. AG1478, a specific EGF receptor kinase inhibitor, abolished the prolonged erk-1 and erk-2 activation produced by EGF in cells pretreated with BAPTA/AM. This indicates that the prolonged activation of erk-1 and erk-2 produced in the presence of BAPTA/AM requires continuous signaling from the EGF receptor kinase.     

Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.     

MEK ablation in MCF-7 cells blocks DNA synthesis induced by serum, but not by estradiol or growth factors

The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.     

The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition

The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.     

Cyclic AMP induces activation of extracellular signal-regulated kinases in HL-60 cells: role in cAMP-induced differentiation

It is well known that elevated intracellular cAMP induces growth arrest and the differentiation of HL-60 cells to neutrophil-like cells. The present study was designed to assess the regulation of the extracellular signal-regulated kinase (ERK) pathway by cAMP and its association with differentiation in HL-60 cells. We found that 8-bromoadenosine-3',5'-cyclic-monophosphate (8Br-cAMP)-induced the activation of ERK and mitogen-activated protein kinase (MEK), but inhibited B-Raf kinase via a protein kinase A (PKA)-mediated mechanism. Prolonged exposure to 8Br-cAMP increased the phorbol 12-myristate 13-acetate (TPA)-stimulated superoxide generation and CD14 expression that characterize the differentiation phenotype, which was blocked by MEK-1 inhibitor. These data suggest that cAMP-induced ERK activation is essential for the differentiation of HL-60 cells, independently of B-Raf.     

Insulin-like growth factor binding protein-2 stimulates proliferation and activates multiple cascades of the mitogen-activated protein kinase pathways in NIH-OVCAR3 human epithelial ovarian cancer cells

Insulin-like growth factor binding protein-2 (IGFBP-2), the second most abundant IGFBP in the circulation, is dramatically increased in the serum and ovarian cyst fluid of women with epithelial ovarian cancer. The specific role of IGFBP-2 in ovarian carcinogenesis remains elusive. Using NIH-OVCAR3 human epithelial ovarian cancer cells, we have evaluated the effects of IGFBP-2 and its antibody on cell proliferation, activation of mitogen-activated protein kinase (MAP kinase) pathways and on cytokine expression. Treatment of the cells with IGFBP-2 stimulates cell growth significantly (p<.05) and potentiates the activation of (1) the extracellular signal regulated kinase (ERK1/2) signaling pathway, which transduces cell-specific growth and differentiation signals; (2) the stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) pathway, which is activated by environmental stresses, inflammatory cytokines, growth factors and G-protein coupled receptor (GPCR) agonists; and (3) the p38 MAP kinase pathway, which mediates inflammatory and stress responses. Suppression of IGFBP-2, with its neutralizing antibody, significantly (p<.05) retards cell growth, blocks the activation of all three cascades of the MAPK pathways and downregulates the expression of a number of potential cancer-promoting cytokines. These novel findings may have important clinical implications for developing innovative strategies for the treatment and management of ovarian cancer.     

Role of notch-1 and E-cadherin in the differential response to calcium in culturing normal versus malignant prostate cells

A panel of expression markers was validated and used to document that, when radical prostatectomy specimens are cultured in low (i.e., <260 micromol/L)-calcium (Ca2+)-serum-free, growth factor-defined (SFD) medium, what grows out are not prostatic cancer cells but basally derived normal transit-amplifying prostatic epithelial cells. The selective outgrowth of the normal transit-amplifying versus prostatic cancer cells is due to the differential effect of low-Ca2+ medium on the structure of Notch-1 and E-cadherin signaling molecules. In low-Ca2+ medium, Notch-1 receptor is conformationally in a constitutively active, cell autonomous form not requiring reciprocal cell-cell (i.e., ligand) interaction for signaling. Such signaling is required for survival of transit-amplifying cells as shown by the death of transit-amplifying cells induced by treatment with a series of chemically distinct gamma-secretase inhibitors to prevent Notch-1 signaling. Conversely, in low-Ca2+ medium, E-cadherin is conformationally inactive preventing cell-cell homotypic interaction, but low cell density nonaggregated transit-amplifying cells still survived because Notch-1 is able to signal cell autonomously. In contrast, when medium Ca2+ is raised to >400 micromol/L, Notch-1 conformationally is no longer constitutively active but requires cell-cell contact for reciprocal binding of Jagged-1 ligands and Notch-1 receptors between adjacent transit-amplifying cells to activate their survival signaling. Such cell-cell contact is enhanced by the elevated Ca2+ inducing an E-cadherin conformation allowing homotypic interaction between transit-amplifying cells. Such Ca(2+)-dependent, E-cadherin-mediated interaction, however, results in cell aggregation, stratification, and inhibition of proliferation of transit-amplifying cells via contact inhibition-induced up-regulation of p27/kip1 protein. In addition, transit-amplifying cells not contacting other cells undergo squamous differentiation into cornified (i.e., 1% SDS insoluble) envelopes and death in the elevated Ca2+ medium. Stratification and contact inhibition induced by elevated Ca2+ are dependent on E-cadherin-mediated homotypic interaction between transit-amplifying cells as shown by their prevention in the presence of a cell-impermanent, E-cadherin neutralizing antibody. In contrast to growth inhibition of normal transit-amplifying cells, supplementation of low-Ca(2+)-SFD medium with 10% FCS and raising the Ca2+ to >600 micromol/L stimulates the growth of all prostate cancer cell lines tested. Additional results document that, at physiologic levels of Ca2+ (i.e., >600 micromol/L), prostatic cancer cells are not contact inhibited by E-cadherin interactions and Notch-1 signaling is no longer required for survival but instead becomes one of multiple signaling pathways for proliferation of prostatic cancer cells. These characteristic changes are consistent with prostate cancer cells' ability to metastasize to bone, a site of high-Ca2+ levels.     

Direct integrin alphavbeta6-ERK binding: implications for tumour growth

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.     

Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal--regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines

The cellular response to cisplatin involves activation of multiple signal transduction pathways, including the mitogen-activated protein (MAP) kinase pathways. In this study, we compared the cisplatin-induced activation of two MAP kinases, c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its derivative cisplatin-resistant cell lines CP70 and C200. Dose-dependent and time-dependent activation of JNK1 and ERK1/2 occurred in each of the three cell lines in response to cisplatin treatment. The requirement of higher concentrations of cisplatin for induction of maximum activation of JNK1 and ERK1/2 was correlated with increased levels of cisplatin resistance. In addition, inhibition of cisplatin-induced ERK activation, using the MAP/ERK kinase 1 synthetic inhibitor PD98059, resulted in enhanced sensitivity to cisplatin in all three cell lines. These results suggest that cisplatin-induced ERK1/2 activity is not responsible for the acquired cisplatin resistance in CP70 and C200 cells but rather provides a general cytoprotective effect in both cisplatin-sensitive and cisplatin-resistant cell lines. In conclusion, different patterns of cisplatin-induced JNK1 and ERK1/2 activation are observed in cell lines with different levels of cisplatin sensitivity, and inhibition of cisplatin-induced ERK1/2 activation enhances sensitivity to cisplatin in both cisplatin-sensitive and cisplatin-resistant cell lines.