Insulin receptor substrate-1 is an important mediator of ovarian cancer cell growth suppression by all-trans retinoic acid

There is a need to identify more effective drugs for the treatment of ovarian cancer as it is the leading cause of death among gynecologic tumors. All-trans retinoic acid (ATRA), a natural retinoid, arrests the growth of CA-OV3 ovarian carcinoma cells in G(0)-G(1). Because the insulin-like growth factor-I receptor has been implicated in the proliferation of various tumors, we investigated its potential role in the suppression of ovarian cancer cell growth by ATRA. Our studies revealed that insulin receptor substrate-1 (IRS-1) protein levels decrease in CA-OV3 cells on ATRA treatment, whereas no differences in IRS-1 levels were seen in the ATRA-resistant SK-OV3 cells. Moreover, CA-OV3 clones overexpressing IRS-1 were growth inhibited less by ATRA, whereas SK-OV3 clones in which levels of IRS-1 were reduced by expression of antisense IRS-1 became sensitive to growth inhibition by ATRA treatment. Studies to determine the mechanism by which ATRA reduced IRS-1 expression showed that ATRA altered steady-state levels of IRS-1 mRNA and the stability of IRS-1 protein. Finally, the role of IRS-1 as a potential molecular target of ATRA in ovarian tumors was assessed by immunohistochemistry in an ovarian cancer tissue array. Compared with normal ovary, the majority of malignant epithelial ovarian tumors overexpressed IRS-1. Thus, there seems to be a correlation between IRS-1 expression and malignancy in ovarian tumors. Our results suggest that IRS-1 is in fact an important growth-regulatory molecule that can be a potential effective target for chemotherapeutic intervention with growth-suppressive agents, including retinoids.     

Cell cycle and transcriptional control of human myeloid leukemic cells by transforming growth factor beta

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.     

Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth

 

The mechanism of suberoylanilide hydroxamic acid in cell growth inhibition involved induction of pRb-2/p130 interaction and nuclear translocation with E2F-4, followed by significant repression in E2F-1 and PCNA nuclear levels, which led to inhibition in DNA synthesis in mammary epithelial cell lines.

Synopsis

Background

Hybrid polar compounds (HPCs) have induced cell growth arrest, terminal differentiation and/or apoptosis in various transformed cell lines. We have previously reported that the prototype HPC (hexamethylene bisacetamide [HMBA]) was able to arrest the growth of transformed mammary (TM) 2H cells (p53 null), a highly tumorigenic mouse mammary epithelial cell line, by inhibiting G1 kinase activities, concomitant with an increase in the cyclin D2 protein level and hypophosphorylated isoforms of the three pRb pocket proteins, which led to the formation of stable cyclin D2/pRb complexes and G1 cell arrest. It has been reported that the second generation of HPCs (suberoylanilide hydroxamic acid [SAHA]), structurally related to but 2000-fold more potent than HMBA, was an inhibitor of histone deacetylase activity and caused accumulation of hyperacetylated histone H4 in murine erythroleukemia.

Objectives

To determine the mechanism of SAHA in cell growth inhibition in TM10 (p53 wt) and TM2H (p53 null) hyperplastic mouse mammary cell lines.

Methods

TM10 and TM2H cells were examined in the presence or absence of 2.5 μM SAHA for cell growth rate by [³H]-thymidine uptake, DNA synthesis by flow cytometry after cells were labeled with BrdU, G1/S cyclin-dependent kinase (cdk) activities, phosphorylation levels of pRb pocket proteins, protein levels of E2F-1, PCNA and p21, pRb-2/p130 interaction, and nuclear localization with E2F-4 by western blot, immunoprecipitation and immunostaining assays.

Results

SAHA was able to arrest cell growth at G1, and inhibited DNA synthesis in both TM10 and TM2H cell lines. Cell growth arrest was accompanied by increases in histone H3 and H4 protein and acetylation levels, a profound increase in the interaction and nuclear localization of pRb-2/p130–E2F-4 complexes, significant reductions in E2F-1 and PCNA protein levels, inhibition in G1/S cdk activities and increases in the levels of hypophosphorylated isoforms of three pRb pocket proteins.

Conclusion

A novel mechanism of SAHA mediated growth inhibition through significant increases in the formation and nuclear localization of pRb-2/p130–E2F-4 complexes, which resulted in cell growth arrest and significant repression in the levels of two key molecules, E2F-1 and PCNA, essential for DNA synthesis in two mouse mammary epithelial cell lines. These responses to SAHA were independent of the p53 status of the cell; however, reversibility of SAHA-mediated growth correlated with the wild type p53 status.     

Retinoic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression

Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor beta (RAR-beta) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-beta in the growth regulation of SCLC by retinoids, we restored RAR-beta expression in RAR-beta-negative H209 SCLC cells by retroviral transduction (H209-RAR-beta). We found that H209-RAR-beta, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-beta cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-beta cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-beta, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-beta cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-beta gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-beta.     

Molecular analysis of selected cell cycle regulatory proteins during aerobic and hypoxic maintenance of human ovarian carcinoma cells.

We have previously reported on the development of an in vitro model system for studying the effect of hypoxia on ovarian carcinoma cell proliferation and invasion (Krtolica and Ludlow, 1996). These data indicate that the cell division cycle is reversibly arrested during the G1 phase. Here, we have continued this study to include the proliferation properties of both aerobic and hypoxic human ovarian carcinoma cells at the molecular level. The growth suppressor product of the retinoblastoma susceptibility gene, pRB, appears to be functional in these cells as determined by SV40 T-antigen binding studies. Additional G1-to-S cell cycle regulatory proteins, cyclins D and E, cyclin-dependent kinases (cdks) 4 and 2, and cdk inhibitors p27 and p18, also appear to be intact based on their apparent molecular weights and cell cycle stage-specific abundance. During hypoxia, there is a decrease in abundance of cyclins D and E, with an increase in p27 abundance. cdk4 activity towards pRB and cdk2 activity towards histone H1 are also decreased. Co-precipitation studies revealed an increased amount of p27 complexing with cyclin E-cdk2 during hypoxia than during aerobic cell growth. In addition, pRB-directed phosphatase activity was found to be greater in hypoxic than aerobic cells. Taken together, a model is suggested to explain hypoxia-induced cell cycle arrest in SKA human ovarian carcinoma cells.     

Expression of pRB, cyclin/cyclin-dependent kinases and E2F1/DP-1 in human tumor lines in cell culture and in xenograft tissues and response to cell cycle agents

Purpose: Cell cycle regulatory components are interesting targets for cancer therapy. Expression of pRb, cyclin D1, cdk4, cyclin E, cdk2, E2F1 and DP-1 was determined in MCF-7 and MDA-MB-468 breast carcinoma cells, H460 and Calu-6 non-small cell lung carcinoma cells, H82 and SW2 small cell lung carcinoma cells, HCT116 and HT29 colon carcinoma cells and LNCaP and DU-145 prostate carcinoma cells. Methods: For Western blotting, the ratio with actin expression was used to normalize the data; all lines were run on the same gels. Results: In cell culture, pRb was not detected in MB-468 and H82 was low in SW2 and DU-145 and highest in HCT116; in tumors, pRb was not detected in MB-468, H82, SW2, and DU-145 and was highest in LNCaP and Calu-6. Cyclin D1 was not detected in SW2 cells in culture, was low in MB-468 and H82, and was highest in LNCaP and H460; in tumors, cyclin D1 was low in MB-468, H460, SW2 and DU145, and was highest in LNCaP. In cell culture, cdk4 was lowest in Calu-6, HCT116, HT29 and DU-145 and highest in H82 and SW2; in tumors, cdk4 was low in MCF-7, MB-468, H460, Calu-6 and HCT116 and was very high in the SW2. Expression of cyclin E was very low in MCF-7 and HT29 and high in H460 in culture and was very low in MCF-7, H460, Calu-6, H82, HT29 and DU-145 in tumors and high in HCT116 and LNCaP. In cell culture, E2F1 was lowest in MB-468, Calu-6, HT29 and DU-145 cells and highest in LNCaP cells; in tumors, E2F1 was lowest in MCF-7, MB-468 and Calu-6 and highest in LNCaP. In cell culture, DP-1 was lowest in MB-468, HCT116 and HT29 and highest in SW2. The MCF-7 and MB-468 lines were most resistant to flavopiridol and olmoucine and the H460 and Calu-6 lines were most resistant to genistein. The SW2 tumor was most responsive to flavopiridol and olomoucine. Conclusions: There is a high degree of variability in the expression of cell cycle components in human tumor cell lines, resulting in complexity in predicting response to cell cycle directed agents. Received: 27 October 1999 / Accepted: 16 March 2000     

Expression of the cell cycle regulator p27(Kip1) in normal squamous epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma of the uterine cervix. Immunohistochemistry and functional aspects of p27(Kip1).

BACKGROUND: Abnormality of cell cycle regulators and tumor suppressors, such as cyclin dependent kinase inhibitors (cdkIs), has been reported in malignant tumors. The current study was undertaken to examine the involvement of a cdkI, p27(Kip1) (p27), in the neoplastic process of the uterine cervical epithelium. METHODS: Immunohistochemical staining of p27 was performed in samples of normal cervical tissue (30 samples), cervical intraepithelial neoplasias (CINs; 17 samples), and invasive squamous cell carcinoma (SCC; 25 samples). The results were compared with the expression levels of Ki-67, cdk2, and cyclin E. The functional aspects of the p27 protein, such as its ability to bind to cdk2 and the phosphorylation activity of p27-bound cdk2, also were evaluated with an immunoprecipitation and histone H1 kinase assay. RESULTS: In normal cervical epithelia, the expression of p27 was strong in the intermediate and superficial cells but very weak in the parabasal cells. In CIN samples, the expression of p27 was negligible. The expression of p27 in these tissues showed an inverse topologic correlation to that of Ki-67, cdk2, and cyclin E. However, it is noteworthy that the number of p27 positive cells increased in SCC samples that also showed increased expression of Ki-67, cdk2, and cyclin E. The p27 protein in SCC samples was bound to cdk2 and cyclin E. However, cdk2 that was bound to p27 still possessed histone H1 kinase activity. CONCLUSIONS: The expression of p27 may be involved in the growth regulation of the normal squamous epithelium in the uterine cervix. However, aberrant function of p27 expression may occur in invasive SCC of the cervix.     

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines.

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1 h, 42 degree C). In IGROV-1 cells, a low concentration (15 microg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27(Kip1) and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

Raf-1 kinase activity predicts for paclitaxel resistance in TP53mut, but not TP53wt human ovarian cancer cells

We have recently reported that there is a significant Raf-1 kinase dependency of paclitaxel resistance in human cervical tumor cell lines. In light of the possibility that Raf-1 kinase inhibitors could be used to enhance paclitaxel responsiveness in ovarian cancer, we have characterized the Raf-1 kinase dependency of paclitaxel resistance in ovarian cancer cells. The relationship between Raf-1 kinase activity and the sensitivity to clinically relevant paclitaxel concentrations was determined in four ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/WT and OAW42/WT). Furthermore, in recognition that such a drug combination would initially be used in patients whose tumors have recurred following cisplatin/paclitaxel treatment, we also determined the Raf-1 kinase dependency of paclitaxel cytotoxicity in cisplatin resistant variants of two of the ovarian cell lines (2780/CP and OAW42/CP). In the two cell lines (2780/WT and OAW42/WT) that possess a wild-type TP53 (TP53wt), the relationship between Raf-1 kinase activity and paclitaxel resistance was different from that observed in the cervical tumor cell lines. In these cell lines, paclitaxel-induced far more cell killing than would have been predicted from their Raf-1 kinase activity. However, in the ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/CP and OAW42/CP) that have a mutant TP53 (TP53mut), the cytotoxicity induced by 60 nM paclitaxel exhibited the same relationship to Raf-1 kinase activity as previously observed in cervical tumor cell lines. These data suggest that the therapeutic efficacy of paclitaxel in ovarian cancer patient whose tumors have TP53mut might be increased if it is administered in combination with Raf-1 kinase inhibitors, e.g., ISIS 5132.     

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.     

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.     

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine?;) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1?h, 42°C). In IGROV-1 cells, a low concentration (15?µg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27^Kip1 and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

Differential retinoblastoma and p16(INK4A) protein expression in neuroendocrine tumors of the lung

BACKGROUND: Neuroendocrine neoplasms of the lung represent a wide spectrum of phenotypically and biologically distinct entities. Their histopathologic diagnosis, which carries therapeutic and prognostic significance, may sometimes be difficult because of their overlapping features. We previously demonstrated that large cell neuroendocrine carcinomas (LCNECs) and small cell lung carcinomas (SCLCs) failed to show positive nuclear staining of RB protein (RB-), whereas typical and atypical carcinoids (TCs and ACs) showed nuclear RB immunostaining (RB+). METHODS: In the current study, a series of 58 surgically resected lung tumors, of which 33 tumors were initially diagnosed as SCLCs and 25 as TCs or ACs, were studied for RB and p16 protein expression by immunohistochemistry. They were also reviewed for their pathologic diagnosis; the reviewers were blinded to the RB and p16 protein status. RESULTS: Nineteen tumors were diagnosed as TCs, 5 as ACs, 7 as LCNECs, and 27 as SCLCs. Three of seven LCNECs were RB+, whereas the other four were RB-. In contrast, all 19 TCs were RB+ and all 27 SCLCs were RB-. In addition, two of five ACs were RB+, whereas the other three were RB-. Interestingly, all 3 RB+ LCNECs and the 1 RB+ AC tested failed to show nuclear staining of p16 protein in any tumor cells (p16-), although some normal stromal cells showed nuclear staining of p16 protein (p16+) as positive internal controls, indicating loss of p16 function in these tumors. It is also noteworthy that the three RB+ LCNECs were initially diagnosed as SCLCs and one of the RB- ACs was initially considered a TC. With the exception of TCs, tumors were significantly more prevalent among heavy smokers with >20 pack-years compared with nonsmokers and light smokers with < or = 20 pack-years (P < 0.01). CONCLUSIONS: These findings suggest that all SCLCs and LCNECs have abnormalities in the p16:RB pathway, as do at least certain ACs, whereas the p16:RB pathway is normal in TCs.