OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC), the leading cause of death from gynecological malignancy, is a poorly understood disease. The typically advanced presentation of EOC with loco-regional dissemination in the peritoneal cavity and the rare incidence of visceral metastases are hallmarks of the disease. These features relate to the biology of the disease, which is a principal determinant of outcome. EOC arises as a result of genetic alterations sustained by the ovarian surface epithelium (OSE; ref. 3). The causes of these changes are unknown but are manifest by activation of oncogenes and inactivation of tumor-suppressor genes (TSGs). Our analysis of loss of heterozygosity at 11q25 identified OPCML (also called OBCAM), a member of the IgLON family of immunoglobulin (Ig) domain-containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules, as a candidate TSG in EOC. OPCML is frequently somatically inactivated in EOC by allele loss and by CpG island methylation. OPCML has functional characteristics consistent with TSG properties both in vitro and in vivo. A somatic missense mutation from an individual with EOC shows clear evidence of loss of function. These findings suggest that OPCML is an excellent candidate for the 11q25 ovarian cancer TSG. This is the first description to our knowledge of the involvement of the IgLON family in cancer.

Higher incidence of isolated brain metastases in ovarian cancer patients with previous early breast cancer.

BACKGROUND: The pathogenesis of brain metastasis as a relatively rare complication of epithelial ovarian cancer is poorly understood. Some observations suggest that brain metastases from ovarian cancer are becoming more common and that ovarian cancers, which metastasize to the brain, may have a different biological pattern. METHODS: Data were extracted from the Edinburgh Ovarian Cancer Database on a cohort of patients managed at the Edinburgh Cancer Centre (UK) between 1998 and 2004. The incidence of brain metastases was compared between patients with previous treatment for early breast cancer and patients without previous treatment for early breast cancer. Baseline characteristics, the time to cancer antigen 125 relapse, the time to brain metastasis, and the radiological pattern of relapse were also compared between these patients. RESULTS: We demonstrate a higher incidence of serous histology (P = 0.02) in patients in remission from early breast cancer and that the incidence of brain metastases in this group is 11.6% compared with 1.1% in patients without prior breast cancer (relative risk = 10.5, P < 0.001). Brain metastases were clinically evident after 45.6 months in patients with previous breast cancer compared with 21 months in patients without previous breast cancer (P = 0.008). Among the patients who developed brain metastases, isolated retroperitoneal lymph node recurrence was noticed in patients in remission from early breast cancer but rarely in other patients. CONCLUSIONS: Ovarian cancer patients with a history of early breast cancer have a higher incidence of brain metastases and a different pattern of disease recurrence. We speculate that a higher incidence of breast cancer early onset mutations in patients with previous early breast cancer underlies these observed differences.

Phase I clinical and pharmacokinetic study of plitidepsin as a 1-hour weekly intravenous infusion in patients with advanced solid tumors.

PURPOSE: Plitidepsin, given as a 1-hour weekly i.v. infusion for 3 consecutive weeks during a 4-week treatment cycle, was investigated in patients with solid tumors to determine the maximum tolerated dose and the recommended dose (RD) using this administration schedule. EXPERIMENTAL DESIGN: Consecutive cohorts of patients with metastatic solid tumors or non-Hodgkin's lymphomas were to be treated at escalating doses of plitidepsin in a conventional phase I study including pharmacokinetic analyses of plitidepsin in plasma, whole blood, and blood cell pellets. RESULTS: Forty-nine patients with solid tumors were enrolled, and 48 were treated with plitidepsin (doses from 0.133 to 3.6 mg/m2/week). Dose-limiting toxicities (defining 3.6 mg/m2/week as the maximum tolerated dose) included myalgia, increased creatine phosphokinase levels, and sustained grade 3/4 increases of hepatic enzyme levels. The RD was established at 3.2 mg/m2/week. The most common toxicities were fatigue, vomiting/nausea, anorexia, injection site reaction, and pain, mostly of mild or moderate severity. Muscular toxicity manifested by mild-moderate myalgia, weakness, and/or creatine phosphokinase elevations occurred in approximately 25% of patients and seemed to be dose related. Transient transaminase elevations were frequent but achieved grade 3 or 4 in only approximately 10% of patients. Plitidepsin lacked significant hematologic toxicity. No complete or partial tumor responses were observed; however, five patients had disease stabilization (including one patient with medullary thyroid carcinoma with an unconfirmed partial response and one patient with renal carcinoma with major tumor shrinkage in lung metastases). Pharmacokinetic results for the RD indicated a long plasma half-life give value (16.8 +/- 7.7 hour) and a high volume of distribution value (525.2 +/- 219.3 L). CONCLUSIONS: The recommended dose for plitidepsin given as a weekly 1-hour schedule was 3.2 mg/m2/week. Muscular and liver toxicity were dose limiting at 3.6 mg/m2/week. Additional evaluation of this dose dense schedule is warranted.

Hormone therapy for epithelial ovarian cancer.

PURPOSE OF REVIEW: Hormonal therapy has been used erratically to treat epithelial ovarian cancer since the mid 1960s; however, there has been little progress until recently in our understanding of which patients might benefit from treatment. Some clarity in the field is now beginning to emerge from recent studies, which have started to define biomarkers within epithelial ovarian cancers that link with hormonal response and thereby indicate which cohort of patients might be amenable to hormonal treatment. RECENT FINDINGS: Oestrogen-deprivation therapy in the form of the aromatase inhibitor letrozole has demonstrated activity in three clinical studies. As only a minority of patients will benefit from hormonal approaches, recent efforts have focussed on the use of molecular markers to help identify which subgroup of patients might respond to treatment. Parallel laboratory studies using primary cancer material for which endocrine response is known and model-based findings have identified components of oestrogen signalling pathways that may help predict outcome. SUMMARY: Use of the antioestrogen letrozole in ovarian cancer has consistently demonstrated a significant level of activity and the larger clinical studies have linked response with oestrogen receptor alpha expression and other biomarkers.

Insulin-like growth factor binding proteins IGFBP3, IGFBP4, and IGFBP5 predict endocrine responsiveness in patients with ovarian cancer.

PURPOSE: This study sought to explore the predictive value of the insulin-like growth factor (IGF) binding proteins (IGFBP) as markers of response in ovarian cancer patients treated with the aromatase inhibitor letrozole. EXPERIMENTAL DESIGN: IGFBP mRNA expression in cell lines was measured by quantitative reverse transcription-PCR and IGFBP protein expression measured in sections from primary tumors of patients treated with letrozole by semiquantitative immunohistochemistry. RESULTS: Quantitative reverse transcription-PCR analysis showed that IGFBP3 and IGFBP5 were down-regulated and IGFBP4 was up-regulated by 17beta-estradiol (E(2)) in an estrogen receptor (ER)-positive ovarian cancer cell line. Expressions of IGFBP1, IGFBP2, and IGFBP6 were unaffected by E(2). The E(2) modulation of these genes was reversed by tamoxifen. Using ERalpha-specific (propyl pyrazole triol) and ERbeta-specific (diarylpropionitrile) agonists, the gene expression modulations produced by E(2) could be replicated by propyl pyrazole triol but not by diarylpropionitrile. For ovarian cancer patients being treated with letrozole, we tested the predictive value of the IGFBPs in paraffin-fixed sections from their primary tumors by semiquantitative immunohistochemistry. Using serum CA125 as an indicator of progression/response, significant differences in expression levels of IGFBPs were observed between tumors from CA125 responding/stable patients compared with tumors from progressing patients. Mean immunoscores for IGFBP3 and IGFBP5 were significantly lower, and mean expression of IGFBP4 was significantly higher in tumors from patients demonstrating CA125 response or stabilization compared with CA125 progression. CONCLUSION: These results indicate that expression levels of certain IGFBP family members in ovarian cancers are estrogen regulated and can, thus, help identify patients who could benefit from endocrine therapy.

Antiestrogen therapy is active in selected ovarian cancer cases: the use of letrozole in estrogen receptor-positive patients.

PURPOSE: To evaluate the efficacy of the aromatase inhibitor letrozole in preselected estrogen receptor (ER)-positive relapsed epithelial ovarian cancer patients and to identify markers that predict endocrine-sensitive disease. EXPERIMENTAL DESIGN: This was a phase II study of letrozole 2.5 mg daily until clinical or marker evidence of disease progression in previously treated ER-positive ovarian cancer patients with a rising CA125 that had progressed according to Rustin's criteria. The primary end point was response according to CA125 and response evaluation criteria in solid tumors (RECIST) criteria. Marker expression was measured by semiquantitative immunohistochemistry in sections from the primary tumor. RESULTS: Of 42 patients evaluable for CA125 response, 7 (17%) had a response (decrease of >50%), and 11 (26%) patients had not progressed (doubling of CA125) following 6 months on treatment. The median time taken to achieve the CA125 nadir was 13 weeks (range 10-36). Of 33 patients evaluable for radiological response, 3 (9%) had a partial remission, and 14 (42%) had stable disease at 12 weeks. Eleven patients (26%) had a PFS of >6 months. Subgroup analysis according to ER revealed CA125 response rates of 0% (immunoscore, 150-199), 12% (200-249), and 33% (250-300); P = 0.028, chi(2) for trend. Expression levels of HER2, insulin-like growth factor binding protein 5, trefoil factor 1, and vimentin were associated with CA125 changes on treatment. CONCLUSIONS: This is the first study of a hormonal agent in a preselected group of ER-positive ovarian cancer patients. A signature of predictive markers, including low HER2 expression, predicts response.

Sensitivity to pertuzumab (2C4) in ovarian cancer models: cross-talk with estrogen receptor signaling.

Pertuzumab (Omnitarg, rhuMab 2C4) is a humanized monoclonal antibody, which inhibits HER2 dimerization. Because it has shown some clinical activity in ovarian cancer, this study sought to identify predictors of response to this agent in a model of ovarian cancer. A panel of 13 ovarian cancer cell lines was treated with heregulin beta1 (HRGbeta1) or transforming growth factor-alpha, and cell proliferation was assessed. Both agents increased cell number in the majority of cell lines studied, the response to both being similar (r = 0.83; P = 0.0004, Pearson test). HRGbeta1 stimulation could be partially reversed by pertuzumab in 6 of 13 cell lines, with complete reversal in PE04 and PE06 cells. Addition of pertuzumab to transforming growth factor-alpha-stimulated cells produced growth inhibition in 3 of 13 cell lines (PE01, PE04, and PE06). The magnitude of HRGbeta1-driven growth stimulation correlated significantly with an increase in extracellular signal-regulated kinase 2 (P = 0.037) but not Akt (P = 0.99) phosphorylation. Such HRGbeta1-driven phosphorylation of extracellular signal-regulated kinase 1/2 and Akt could be reduced with pertuzumab, accompanied by changes in cell cycle distribution. In cell lines responsive to pertuzumab, HRGbeta1-enhanced phosphorylation of HER2 (Tyr(877)) was reduced. Estrogen-stimulated changes in growth, cell cycle distribution, and signaling were reversed by pertuzumab, indicating cross-talk between HER2 and estrogen signaling. These data indicate that there is a subset of ovarian cancer cell lines sensitive to pertuzumab and suggest possible predictors of response to identify patients who could benefit from this therapy. Furthermore, we have identified an interaction between HER2 and estrogen signaling in this disease.

Altered ErbB receptor signaling and gene expression in cisplatin-resistant ovarian cancer.

The majority of ovarian cancer patients are treated with platinum-based chemotherapy, but the emergence of resistance to such chemotherapy severely limits its overall effectiveness. We have shown that development of resistance to this treatment can modify cell signaling responses in a model system wherein cisplatin treatment has altered cell responsiveness to ligands of the erbB receptor family. A cisplatin-resistant ovarian carcinoma cell line PE01CDDP was derived from the parent PE01 line by exposure to increasing concentrations of cisplatin, eventually obtaining a 20-fold level of resistance. Whereas PE01 cells were growth stimulated by the erbB receptor-activating ligands, such as transforming growth factor-alpha (TGFalpha), NRG1alpha, and NRG1beta, the PE01CDDP line was growth inhibited by TGFalpha and NRG1beta but unaffected by NRG1alpha. TGFalpha increased apoptosis in PE01CDDP cells but decreased apoptosis in PE01 cells. Differences in extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling were also found, which may be implicated in the altered cell response to ligands. Microarray analysis revealed 51 genes whose mRNA increased by at least 2-fold in PE01CDDP cells relative to PE01 (including FRA1, ETV4, MCM2, AXL, MT3, TRAP1, and FANCG), whereas 36 genes (including IGFBP3, TRAM1, and KRT4 and KRT19) decreased by a similar amount. Differential display reverse transcriptase-PCR identified altered mRNA expression for TCP1, SLP1, proliferating cell nuclear antigen, and ZXDA. Small interfering RNA inhibition of FRA1, TCP1, and MCM2 expression was associated with reduced growth and FRA1 inhibition with enhanced cisplatin sensitivity. Altered expression of these genes by cytotoxic exposure may provide survival advantages to cells including deregulation of signaling pathways, which may be critical in the development of drug resistance.

The IgLON family in epithelial ovarian cancer: expression profiles and clinicopathologic correlates.

PURPOSE: The IgLON family of cell adhesion molecules, comprising OPCML, HNT, LSAMP, and NEGR1, has recently been linked to cancer, through two of its members being proposed as tumor suppressors. We examined the expression profile of the family in human sporadic epithelial ovarian cancer and the normal ovary. EXPERIMENTAL DESIGN: We determined the expression level of each IgLON in a panel comprising 57 tumor and 11 normal ovarian samples by quantitative real-time reverse transcription-PCR. The results were statistically tested for associations with clinicopathologic variables. RESULTS: OPCML, LSAMP and NEGR1 exhibited reduced expression in the tumor samples relative to the normal samples, whereas HNT expression was elevated. Statistically significant changes were specific to histologic type. The expression levels of individual IgLONs were correlated, the most significant finding being a positive correlation between LSAMP and NEGR1. LSAMP expression was also negatively correlated with overall survival and was found to be a negative predictor of outcome. CONCLUSIONS: The expression of the IgLON family is altered in sporadic epithelial ovarian tumors in comparison to the normal ovary. In our small but representative cohort of patients, we have found significant correlations and associations in expression and clinicopathology that suggest a wider role of the family in ovarian cancer.

Glucuronidation as a mechanism of intrinsic drug resistance in human colon cancer: reversal of resistance by food additives.

Colon cancer exhibits inherent insensitivity to chemotherapy by mechanisms that are poorly characterized. We have shown that human colon cancer cells are efficient in drug conjugation catalyzed by UDP-glucuronosyltransferases (UGTs) and now report on the role of glucuronidation in de novo resistance to two topoisomerase I inhibitors. Identification of the UGT responsible for glucuronidation of SN-38 and the anthraquinone NU/ICRF 505 was achieved by first using a panel of human cDNA-expressed isozymes to measure conjugating activity. HT29 colon cancer cells were then probed by reverse transcriptase-PCR, Western Blot analysis, and liquid chromatography with mass spectrometry for their profile and activity of UGT isozymes and screened for effective inhibitors of glucuronidation. Expression analysis was also conducted in colon cancer biopsies and paired adjacent normal colon specimens. UGT1A9 was identified as the isozyme catalyzing biotransformation of the two compounds in HT29 cells and propofol as an effective competitive inhibitor of this metabolism. Inhibition of glucuronidation resulted in up to a 5-fold enhancement in drug activity. The majority of colon cancer biopsies studies expressed UGT protein at levels greater than in HT29 cells but with marked interpatient variations and proficiently glucuronidated the two anticancer drugs. A range of UGT aglycones were capable of modulating glucuronidation in the biopies with octylgallate being 10-fold more potent (ID(50) 24 microM) than propofol. In a subset of tumors (33%), UGT protein levels and activity exceeded that of paired normal colon. Glucuronidation may represent a mechanism of intrinsic drug resistance in colon cancer open to modulation by a range of food additives and proprietary medicines.

Identification of clinically relevant genes on chromosome 11 in a functional model of ovarian cancer tumor suppression.

Microcell-mediated transfer of normal chromosome 11 (chr 11) to a clonal derivative of the ovarian cancer cell line, OVCAR3, was performed and generated independent hybrids with a common set of phenotypes: inhibition of cell growth and of cellular migration in vitro; and inhibition of tumor growth in vivo. Differential display reverse transcriptase-PCR (RT-PCR), cDNA-representational difference analysis, and hybridization of cDNA high-density filter arrays identified altered mRNAs associated with these phenotypic alterations. Quantitative RT-PCR-based validation of each altered mRNA eliminated false positives to identify a reduced set of expression differences. Twelve products were confirmed as up-regulated and 4 as down-regulated upon introduction of chr 11. Strikingly, 4 of the 12 up-regulated genes were located on chr 11. Expression analysis of selected products by quantitative RT-PCR in a series of 18 human primary ovarian tumors revealed several associations with clinicopathological features. Importantly, low expression of two products, the lysosomal protease CTSD and the lens crystallin CRYAB, was significantly associated with adverse patient survival. Immunohistochemical analysis of CTSD in a larger independent panel of 58 primary ovarian tumors confirmed that low CTSD was associated with poor survival. Furthermore, low CTSD was significantly associated with serous histology and advanced tumor stage. The combined approach of microcell-mediated chromosome transfer and expression difference analysis has identified several altered mRNAs in a model of chr 11-mediated ovarian tumor suppression. The detailed contextual characterization of these genes will determine the extent of their involvement in neoplastic development.

Estrogen receptor-alpha mediates gene expression changes and growth response in ovarian cancer cells exposed to estrogen.

Estrogens play a significant role in the development, growth, invasion and metastasis of ovarian tumors. The transcriptional program regulated by 17beta-estradiol (E(2)) in human ovarian cancer cell lines was analyzed using cDNA microarrays containing 1200 cancer-related genes. Twenty-eight transcripts had at least a threefold change in expression in E(2)-treated PEO1 ovarian carcinoma cells compared with controls. These differences were confirmed by real-time quantitative PCR and shown to be dependent upon the expression of functional estrogen receptor-alpha (ERalpha). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ERalpha- and ERbeta-specific ligands allowed molecular dissection of the E(2) response and showed that ERalpha activation was responsible for the observed changes in gene expression, whereas ERbeta played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E(2). Actinomycin D was used to show that changes in gene expression levels induced by E(2) were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ERalpha-mediated, and not ERbeta-mediated, transcription.

Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer.

PURPOSE: We sought to identify determinants of growth response to the Raf-1-targeted antisense oligonucleotide (ASO; ISIS 5132) using a large panel of ovarian cancer cell lines. EXPERIMENTAL DESIGN: First-(ISIS 5132) and second-generation (ISIS 13650) anti-Raf 1 ASOs were compared with control oligonucleotides. Growth was assessed by cell counts; apoptosis was assessed by poly(ADP-ribose) polymerase cleavage; and cell cycle analysis was assessed by flow cytometry. Protein expression was detected by Western blot analysis, and mRNA expression was detected by quantitative reverse transcription-PCR. Raf-1 kinase activity was detected by anti-Raf-1 immunoprecipitation, followed by myelin basic protein phosphorylation. RESULTS: A panel of 15 ovarian cancer cell lines was used to model a range of growth responses to ASOs targeting Raf-1 mRNA. Growth inhibition varied from 10% to >90% inhibition. Growth inhibition was associated with increased apoptosis and accumulation of cells in the G(2)-M and S phases of the cell cycle. Growth response was not related to level of Raf-1 protein expression, Raf-1 kinase activity, intracellular ASO uptake, or degree of Raf-1 protein inhibition. However, ASO growth response was associated with a high proportion of Raf-1 mRNA [relative to total (i.e., Raf-1 + A-Raf + B-Raf) Raf mRNA] and significantly higher Raf-1 kinase activity induction following growth factor (transforming growth factor alpha) stimulation in the cell lines consistent with dependency of these cell lines on Raf-1. CONCLUSIONS: These data indicate that ovarian cancers demonstrate differential sensitivity to ASOs targeted against Raf-1, and target expression levels and degree of utilization of Raf-1 signaling are implicated. Clinically sensitive tumors could feasibly be identified.

CA125 response is associated with estrogen receptor expression in a phase II trial of letrozole in ovarian cancer: identification of an endocrine-sensitive subgroup.

PURPOSE: This study was an open-label Phase II trial of the aromatase inhibitor letrozole (Femara) in patients with relapsed ovarian cancer with evaluation of possible biological markers for response. EXPERIMENTAL DESIGN: 60 patients were treated with letrozole (2.5 mg daily) at the time of CA125 relapse. Disease response was assessed by Union International Contre Cancer (UICC) criteria and by CA125 measurement. Estrogen receptor (ER), progesterone receptor, epidermal growth factor receptor, erbB2, and HSP27 were measured by immunohistochemistry in paraffin-fixed material obtained from the primary tumors at initial surgery. RESULTS: 50 patients were evaluable by UICC criteria, and although no complete or partial responses were obtained, 10 patients had stable disease on scan for at least 12 weeks. CA125 responses were evaluable in 54 patients. A partial marker response (>50% decrease) was seen in 5, and the marker remained stable in an additional 14 patients (25% increase). Tumors from the UICC stable disease group had significantly higher ER (P = 0.027) and progesterone receptor (P = 0.0066) values than the progressive disease group, and a combination of these was strongly associated with stable disease (P < 0.0001). Using CA125 criteria, comparison of the CA125 stable/responding disease with progressive disease indicated that tumors with higher ER (P = 0.013), lower erbB2 (P = 0.026), and higher epidermal growth factor receptor (P = 0.009) were associated with CA125 stable/responsive disease. CONCLUSIONS: These results imply that letrozole treatment can produce disease stabilization and CA125 responses that in turn are linked to higher levels of ER expression. These data suggest the presence of an endocrine-sensitive group that could be targeted in future studies.

Neuregulin expression, function, and signaling in human ovarian cancer cells.

PURPOSE: To investigate the expression and function of neuregulin (NRG) isoforms in ovarian cancer cell lines and tumor samples. EXPERIMENTAL DESIGN: Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry and mRNA by RT-PCR. erbB receptor levels and downstream signaling proteins were measured by Western blot analysis. RESULTS: Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry in 46 of 53 (87%) and 41 of 53 (77%) ovarian carcinomas, respectively. Serous carcinomas express higher levels of NRG-1alpha than endometrioid carcinomas (P = 0.017). NRG mRNA was detected by RT-PCR in 20 of 24 (83%) of ovarian carcinomas and eight of nine (89%) ovarian cancer cell lines. NRG-1alpha stimulated the growth of 5 of 14 cell lines whereas NRG-1beta stimulated 7 of 14 cell lines. The magnitude of NRG growth response was significantly associated with erbB2 expression levels. NRG-1alpha and -1beta (1 nM) growth-stimulated cell lines PE01 and PE06 demonstrated increased tyrosine phosphorylation of erbB2 and elevated tyrosine phosphorylation of ERK1 and ERK2. In contrast, the SKOV-3 cell line, the growth of which was unaffected, did not show these downstream responses. An anti-erbB3 receptor antibody (clone H3.105.5) blocked NRG-1beta growth changes and signaling in these cell lines. Conversely, the anti-erbB4 antibody (clone H4.72.8) enhanced NRG-beta1 growth stimulation. Herceptin also inhibited growth. CONCLUSIONS: With NRG expression in the majority of ovarian carcinomas and cell lines, there is the potential for autocrine regulation of cell growth. Interfering with ligand-receptor interactions by receptor blocking antibodies suggests erbB3 is primarily involved in NRG-1beta-induced proliferation, with erbB4 having a more complex role.

Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence to apoptosis in colorectal cancer cells, enhancing global cytotoxicity.

PURPOSE: To identify determinants of the effect of antisense-mediated Bcl-xl down-regulation (Bcl-xl knockdown) on the response of colorectal cancer cells to SN38, the active metabolite of irinotecan, a topoisomerase I inhibitor licensed for colorectal cancer chemotherapy. EXPERIMENTAL DESIGN: Using wild-type HCT116, p53 null, Bax null, or p21/WAF1 null isogenic derivatives, we measured expression of regulators of cellular response, and associated growth arrests or apoptosis, after SN38 treatment, with or without antisense-mediated Bcl-xl knockdown. RESULTS: A modified phosphorothioate antisense oligonucleotide (ISIS15999) reduced Bcl-xl protein expression by approximately 90%. SN38 induced p53, Bax, Bcl-xl, and p53-dependent p21/WAF1 protein accumulation. The Bax:Bcl-xl ratio changed little. In wild-type HCT116, but not in Bax null cells, Bcl-xl knockdown induced a shift in response from drug-induced senescence to apoptosis, and enhanced the global cytotoxicity of SN38. In p53 null or p21/WAF1 null cells marked apoptosis occurred after SN38 alone, and was additionally enhanced by Bcl-xl knockdown in p21/WAF1 null cells but not in p53 null cells. CONCLUSIONS: Drug-induced senescence is associated with late relapse after therapy in transgenic models of cancer in vivo. We have shown that abolition of p21/WAF1-mediated drug-induced senescence or antisense-mediated Bcl-xl knockdown can both, independently, enhance the apoptotic response of colorectal cancer cells to SN38 in vitro. The growth arrest suppresses a p53-independent apoptotic pathway, whereas Bcl-xl induction suppresses a p53 and Bax-dependent apoptotic pathway. The combination of irinotecan and Bcl-xL antisense merits testing in models of colorectal cancer in vivo.

Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters.

PURPOSE: Matriptase is a type II transmembrane serine protease expressed by cells of surface epithelial origin, including epithelial ovarian tumor cells. Matriptase cleaves and activates proteins implicated in the progression of ovarian cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine the expression of matriptase, and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), in epithelial ovarian cancer and to assign clinicopathological correlations. EXPERIMENTAL DESIGN: We have determined by immunohistochemistry the expression of matriptase and HAI-1 in 54 epithelial ovarian cancers. Statistical analyses of immunohistochemistry expression data with clinical outcome and clinicopathological parameters were then performed. RESULTS: Of 54 tumors tested, 39 (72%) and 11 (20%) were positive for matriptase and for HAI-1, respectively. All HAI-1-positive tumors were also matriptase positive. Analysis of clinicopathological parameters demonstrated a loss of matriptase associated with stage III/IV tumors as compared with stage I/II tumors (P = 0.030). There was also a loss of HAI-1 expression associated with stage III/IV tumors (P = 0.039). Of 34 stage I/II tumors, 28 (82%) stained positive for matriptase, and 10 (29%) stained positive for HAI-1; 10 (29%) tumors showed coexpression. Of 20 stage III/IV tumors, however, 11 stained positive for matriptase (55%), only 1 of which coexpressed HAI-1 (P = 0.039). CONCLUSIONS: Advanced-stage ovarian tumors that express matriptase are more likely to do so in the absence of its inhibitor, HAI-1, indicating that an imbalance in the matriptase:HAI-1 ratio could be important in the development of advanced disease. Such an imbalance could promote the proteolytic activity of matriptase and, consequently, a more invasive phenotype.

Enhanced oxaliplatin-induced apoptosis following antisense Bcl-xl down-regulation is p53 and Bax dependent: Genetic evidence for specificity of the antisense effect.

INTRODUCTION: Oxaliplatin, licensed for colorectal cancer chemotherapy, damages DNA by generating intrastrand and interstrand cross-links and can induce apoptosis via a Bax-dependent pathway. Bcl-xl, an antiapoptotic Bcl-2 family member, regulates apoptosis and chemoresistance in several cancer models. Bcl-xl expression correlates with invasiveness in primary colorectal cancer. Bcl-xl may therefore represent a therapeutic target in this disease. We used the mismatch repair-deficient HCT116 colorectal cancer cell line (wild-type HCT116) and p53 null, Bax null, or p21/WAF1 null derivatives to identify genetic determinants of the response to oxaliplatin and tested the hypothesis that antisense-mediated Bcl-xl down-regulation would enhance the apoptotic response in a p53- or Bax-dependent manner. RESULTS: At clinically relevant concentrations, oxaliplatin induced p53 and p53-dependent Bax, Bcl-xl, and p21/WAF1 protein accumulation. A minor degree of apoptosis resulted via a p53- and Bax-dependent pathway. The major response was a transient mixed G(1) and G(2) growth arrest. The G(1) arrest was p53 and p21/WAF1 dependent. A 2'-O-ribose methoxyethyl phosphorothioate antisense oligonucleotide reduced Bcl-xl protein expression by approximately 90% in HCT116 (Bcl-xl knockdown). Missense controls were inactive. Prior Bcl-xl knockdown enhanced the apoptotic and the global cytotoxic effect of oxaliplatin. The extent of enhancement of apoptosis depended on the integrity of the p53- and Bax-mediated apoptotic pathway, providing genetic evidence that the desired proapoptotic antisense effect is due to specific down-regulation of the Bcl-xl target. CONCLUSIONS: The combination of oxaliplatin and Bcl-xl antisense merits testing in models of colorectal cancer in vivo.

Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins.

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.

Enhanced clearance of topoisomerase I inhibitors from human colon cancer cells by glucuronidation.

As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC.