Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis.

Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.

BACKGROUND: KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway. PATIENTS AND METHODS: Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out. RESULTS: From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3-3.7] for erlotinib alone and 2.1 months (95% CI 1.8-5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib. CONCLUSIONS: This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

Tumor-intrinsic and tumor-extrinsic factors impacting hsp90- targeted therapy.

In 1994 the first heat shock protein 90 (Hsp90) inhibitor was identified and Hsp90 was reported to be a target for anticancer therapeutics. In the past 18 years there have been 17 distinct Hsp90 inhibitors entered into clinical trial, and the small molecule Hsp90 inhibitors have been highly valuable as probes of the role of Hsp90 and its client proteins in cancer. Although no Hsp90 inhibitor has achieved regulatory approval, recently there has been significant progress in Hsp90 inhibitor clinical development, and in the past year RECIST responses have been documented in HER2-positive breast cancer and EML4-ALK-positive non-small cell lung cancer. All of the clinical Hsp90 inhibitors studied to date are specific in their target, i.e. they bind exclusively to Hsp90 and two related heat shock proteins. However, Hsp90 inhibitors are markedly pleiotropic, causing degradation of over 200 client proteins and impacting critical multiprotein complexes. Furthermore, it has only recently been appreciated that Hsp90 inhibitors can, paradoxically, cause transient activation of the protein kinase clients they are chaperoning, resulting in initiation of signal transduction and significant physiological events in both tumor and tumor microenvironment. An additional area of recent progress in Hsp90 research is in studies of the posttranslational modifications of Hsp90 itself and Hsp90 co-chaperone proteins. Together, a picture is emerging in which the impact of Hsp90 inhibitors is shaped by the tumor intracellular and extracellular milieu, and in which Hsp90 inhibitors impact tumor and host on a microenvironmental and systems level. Here we review the tumor intrinsic and extrinsic factors that impact the efficacy of small molecules engaging the Hsp90 chaperone machine.

Update on Hsp90 inhibitors in clinical trial.

Twenty-five years ago the first small molecule inhibitors of Hsp90 were identified. In the intervening years there has been dramatic progress in basic scientific understanding of the Hsp90 chaperone machinery and in the role of Hsp90 in malignancy. The first-in-class Hsp90 inhibitor 17-AAG entered into Phase I clinical trials in 1999. There are now 13 Hsp90 inhibitors in clinical trial, representing multiple drug classes, and hundreds of patients have been treated in adult oncology and pediatric oncology trials. This review will provide an overview of the clinical trial results thus far. In addition, pivotal issues in further development of Hsp90 inhibitors as anticancer drugs will be discussed.

FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and transcriptional activity in acute myeloid leukemia cells.

Deregulated accumulation of nuclear beta-catenin enhances transcription of beta-catenin target genes and promotes malignant transformation. Recently, acute myeloid leukemia (AML) cells with activating mutations of FMS-like tyrosine kinase-3 (FLT3) were reported to display elevated beta-catenin-dependent nuclear signaling. Tyrosine phosphorylation of beta-catenin has been shown to promote its nuclear localization. Here, we examined the causal relationship between FLT3 activity and beta-catenin nuclear localization. Compared to cells with wild-type FLT3 (FLT3-WT), cells with the FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain mutation (FLT3-TKD) had elevated levels of tyrosine-phosphorylated beta-catenin. Although beta-catenin was localized mainly in the cytoplasm in FLT3-WT cells, it was primarily nuclear in FLT3-ITD cells. Treatment with FLT3 kinase inhibitors or FLT3 silencing with RNAi decreased beta-catenin tyrosine phosphorylation and nuclear localization. Conversely, treatment of FLT3-WT cells with FLT3 ligand increased tyrosine phosphorylation and nuclear accumulation of beta-catenin. Endogenous beta-catenin co-immunoprecipitated with endogenous activated FLT3, and recombinant activated FLT3 directly phosphorylated recombinant beta-catenin. Finally, FLT3 inhibitor decreased tyrosine phosphorylation of beta-catenin in leukemia cells obtained from FLT3-ITD-positive AML patients. These data demonstrate that FLT3 activation induces beta-catenin tyrosine phosphorylation and nuclear localization, and thus suggest a mechanism for the association of FLT3 activation and beta-catenin oncogeneic signaling in AML.

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling.

Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.

Impact of extracellular folate levels on global gene expression.

Methylation of DNA is associated with gene silencing. DNA methylation uses S-adenosylmethionine (SAM) as the methyl donor and the formation of SAM requires a continuous supply of folate from the extracellular milieu. Low extracellular folate levels are known to result in induction of expression of the human alpha folate receptor in nasopharyngeal epidermoid carcinoma cells. Low folate levels have been implicated in global activation of gene expression. We have investigated the impact of lowering the level of extracellular folate by performing cDNA microarray analysis of global gene expression in human nasopharyngeal carcinoma KB cells grown in folate-deplete and folate-replete medium. We found that expression of only eight genes reproducibly responded to variation of folate levels. Among those, three were up-regulated and five were down-regulated. Examination of one gene, H-cadherin, demonstrated down-regulation in response to folate depletion. Despite the low level of extracellular folate, there was hypermethylation of H-cadherin 5' sequences. These data indicate that low extracellular folate positively and negatively influences the expression levels of a small cohort of genes. The data suggest that folate deficiency is associated with gene-specific methylation/demethylation, rather than global DNA demethylation and transcriptional activation.

The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic chemotherapy.

The Bcr-Abl fusion protein drives leukemogenesis and can render leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90-associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most cytotoxic drugs, but were found to be sensitive to GA. Furthermore, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic effects of DOX in parental HL60 cells. These results indicate that sensitization of Bcr-Abl-expressing cells, but not desensitization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, GA differentially affects leukemia cells depending on their Bcr-Abl expression and selectively increases apoptosis in Bcr-Abl-expressing cells.

Transcriptional activation of p21(WAF1/CIP1) by apicidin, a novel histone deacetylase inhibitor.

Apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-l-2-amino-8-oxodecanoyl)], a novel histone deacetylase inhibitor, has been identified as an antiprotozoal and antiproliferative agent. In this study, we show apicidin induces transcriptional activation of p21(WAF1/CIP1) (p21) in human prostate carcinoma cells. Apicidin induces expression of p21 protein and mRNA and activation of p21 promoter-luciferase reporter constructs. Apicidin causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin. Chromatin immunoprecipitation shows p21 promoter DNA is associated with hyperacetylated histones H3 and H4 after treatment with apicidin. Therefore, the data here demonstrate that apicidin activates p21 transcription associated with the acetylation of histones H3 and H4.

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition.

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Lovastatin induces apoptosis in a primitive neuroectodermal tumor cell line in association with RB down-regulation and loss of the G1 checkpoint.

To develop a new approach to the treatment of primitive neuroectodermal tumors we evaluated the effect of the HMG-CoA reductase inhibitor lovastatin on the Ewing's sarcoma cell line CHP-100. Lovastatin induced neural morphology and markers including neuron-specific enolase and neurofilament protein. The acquisition of neural morphology required new mRNA synthesis, and cDNA microarray analysis confirmed that lovastatin altered the program of gene expression. After morphologic differentiation the cells underwent rapidly progressive apoptosis. In normal development of neuronal progenitors, differentiation signals trigger p21WAF1 accumulation, RB hypophosphorylation, enhanced RB-E2F-1 association, and G1 arrest, and these events have been shown to protect from apoptosis. In contrast, in the Ewing's sarcoma cells lovastatin triggered differentiation without causing cell cycle arrest: p21WAF1 was not induced, RB remained hyperphosphorylated, and RB protein expression and RB-E2F-1 association were markedly downregulated, suggesting that loss of an RB-regulated G1 checkpoint promoted apoptosis. Consistent with this hypothesis, adenoviral p21WAF1 decreased DNA synthesis and partially protected from lovastatin-induced cytotoxicity. The data demonstrate a new model for examining the genetic regulation of cell fate in a neural progenitor tumor and suggest a new approach to the treatment of this neoplasm.

Regulation of BRCA1 by protein degradation.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.

Cyclic AMP induces inhibition of cyclin A expression and growth arrest in human hepatoma cells.

Classical cytotoxic therapy has been minimally useful in the treatment of hepatocellular carcinoma. In an effort to develop a new approach to the treatment of this neoplasm, we have investigated the signal transduction pathways regulating the growth of human hepatoma cells. In the data reported here, cyclic AMP (cAMP), a negative growth regulator for many cells of epithelial origin, induced G1 synchronization and apoptosis in the HepG2 human hepatoma cell line. The effects of cAMP on the components of the G1/S transition were analyzed. There was no detectable effect of two different cAMP analogs, 8-bromo cAMP or dibutyryl cAMP on the level of the D-type cyclins, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, p53, or the cyclin-dependent kinase inhibitors p21 or p27. In contrast, the cAMP analogs induced a dramatic downregulation of cyclin A protein, cyclin A messenger RNA, and cyclin A-dependent kinase activity. Cyclin A-dependent kinase has been shown to be required for the G1-S transition. Furthermore, cyclin A deregulation has been implicated in the pathogenesis of hepatocellular carcinoma. The data reported here suggest a novel signal transduction-based approach to hepatoma therapy.

Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Correlation of expression of bombesin-like peptides and receptors with growth inhibition by an anti-bombesin antibody in small-cell lung cancer cell lines.

The murine anti-bombesin monoclonal antibody, 2A11, has been demonstrated to inhibit growth of some small-cell lung cancer (SCLC) cells in nude mice xenografts and in a clinical trial. To determine if the expression of bombesin-like peptides (BLP) and their receptors (GRP-R and NMB-R) correlate with an in vitro response to 2A11, we measured these parameters in seven SCLC cell lines. Gastrin releasing peptide (GRP) mRNA was detected in three of seven cell lines (NCI-H69, NCI-H345, NCI-H510) and neuromedin B (NMB) mRNA was detected in all seven lines using an RNase protection assay (RPA). Immunoreactive BLP was detected in the cell pellets of all lines (range 0.11-59.90 pmol/mg protein) by a solid phase GRP radioimmunoassay (RIA) using 125I-labeled 2A11. RPA detected GRP-receptor mRNA in two cell lines (NCI-H69 and NCI-H345) and NMB-receptor in three lines (NCI-H345, NCI-H510, and NCI-H660). Reverse transcriptase-PCR confirmed the presence of receptor mRNA in these lines and detected NMB-receptor in an additional three lines (NCI-H69, NCI-H82, and NCI-H187). Calcium mobilization in response to BLP stimulation was detected in the six cell lines expressing either GRP-R or NMB-R mRNA but not in NCI-N417, which had no detectable BLP-receptor. 2A11 (5 microg/ml) inhibited colony formation by 26-61% after 2 weeks in all cell lines except NCI-N417. Thus, growth inhibition by 2A11 requires the presence of at least one BLP-receptor. These findings may be useful in selecting patients with SCLC for treatment with 2A11.

Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(WAF1/CIP1) in human prostate carcinoma cells.

Progression through the cell cycle is controlled by the induction of cyclins and the activation of cognate cyclin-dependent kinases. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor lovastatin induces growth arrest and cell death in certain cancer cell types. We have pursued the mechanism of growth arrest in PC-3-M cells, a p53-null human prostate carcinoma cell line. Lovastatin treatment increased protein and mRNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), increased binding of p21 with Cdk2, markedly inhibited cyclin E- and Cdk2-associated phosphorylation of histone H1 or GST-retinoblastoma protein, enhanced binding of the retinoblastoma protein to the transcription factor E2F-1 in vivo, and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the lovastatin-responsive element was mapped to a region between -93 and -64 relative to the transcription start site. Promoter mutation analysis indicated that the lovastatin-responsive site coincided with the previously identified transforming growth factor-beta-responsive element. These data indicate that in human prostate carcinoma cells an inhibitor of the HMG-CoA reductase pathway can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21.

Regulation of cyclin D1 by calpain protease.

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3.

The propensity of a cell to undergo apoptosis has been proposed to be a determinant for chemotherapy sensitivity that is not directly dependent on specific drug-target interactions. Androgen-independent prostate cancer is typically refractory to cytotoxic drugs, and we tested whether this is due to a loss of the ability to undergo apoptosis. Exposure of the hormone-insensitive and p53-negative human prostate carcinoma cell line PC-3 to 22 microM cisplatin, 1 microM camptothecin, 10 microM tenoposide, 135 nM vincristine, or 10 microM lovastatin for 72 h caused cell death, internucleosomal DNA fragmentation, and morphological changes typical for apoptosis. One microM cycloheximide prevented anticancer drug-induced apoptosis, whereas high concentration (1 mM) of cycloheximide alone induced apoptosis, indicating that protein synthesis was not needed for these cells to undergo apoptosis. Since cycloheximide affected DNA synthesis and proliferation of PC-3 cells, we tested whether the DNA polymerase inhibitor aphidicolin could also suppress drug-induced apoptosis. In contrast to cycloheximide, aphidicolin inhibited only vincristine-induced apoptosis. Cycloheximide prevented drug-induced changes in cell cycle distribution except for vincristine, while aphidicolin led to an accumulation of cells at the G1-S border independent of the drug used. These data indicate that macromolecular synthesis, active cell cycling, and p53 expression are not required for apoptosis to proceed in this system.