The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Efficacy and patient acceptability of the continence dish.

INTRODUCTION AND HYPOTHESIS: The continence dish has been a treatment option since 2002 for women with stress urinary incontinence (SUI) who decline surgery, but few quantitative objective efficacy data are published. We aimed to determine the efficacy and acceptability of this device for pure SUI or mixed incontinence (MUI). METHODS: Prospective interventional cohort study of 100 women with SUI or stress-predominant MUI who were interested to use the device; International Consultation on Incontinence Questionnaire (ICIQ) was primary outcome measure; 24-h pad test and Incontinence Impact Questionnaire (IIQ) were secondary outcomes. Acceptability was determined by device retention for 4 weeks, adverse events and ability to self-insert the device. RESULTS: Of 100 suitable women, 9 were not actually fitted, and 27 did not complete (acceptability: 64/100). The rate of adverse events was 7.7%, with 62.5% of users able to self-insert the device: 22 (34%) had pure SUI; 66% had MUI. In SUI, 68% were 'dry' on ICIQ median value 4.0 (IQR 2.5-8.5); 88% were dry on 24-h pad test (median 0.0, IQR 0.0-8.5). The "dry rate" was lower in MUI: 36% for ICIQ (median 9.0, IQR 5.0-15.0) and 62% for 24-h pad test (median 6.2, IQR 0.95-19.7). A "good" response on IIQ occurred in 88% of SUI and 69% of MUI. CONCLUSION: These new data showing strong objective benefits of the continence dish should be further validated by randomized trials, but this information should be made available to women seeking treatment options for SUI/MUI (particularly in view of concerns regarding mesh mid-urethral slings).

Effect of antibiotics on urine leakage in women with refractory detrusor overactivity: A phase IIb randomized trial.

AIM: Because bacterial cystitis is common in women with refractory detrusor overactivity, the aim was to compare the efficacy of 6 weeks of rotating antibiotics versus placebo, in conjunction with an anticholinergic, in controlling the symptoms of urge incontinence. METHODS: In a multicenter phase IIb double-blinded randomized placebo-controlled trial, women with urodynamically proven refractory detrusor overactivity were randomized in a 2:1 ratio of antibiotics versus placebo for 6 weeks, in addition to darifenacin for 6 months. Any woman with disabling cystitis symptoms was given appropriate antibiotics ("clinical override"). The primary outcome was the degree of urge incontinence change at 6 weeks and 6 months on 24-h pad test. Secondary outcomes were changes in leaks and voids per day measured on 3-day bladder diary and quality of life measures. Microbiological data were collected at all visits. RESULTS: Although 278 women were screened, only 36 were randomized and 33 (91.7%) completed the trial. Leakage on 24-h pad test decreased at 6 months by 75 g in patients receiving antibiotics versus 35 g in placebo. Cure of urge incontinence occurred at 6 months in 10/21 (48%) of antibiotics versus 2/12 (17%) of placebo. Clinical override, necessitating treatment of cystitis, occurred in 41.6% of placebo versus 16.7% of the antibiotic group by 6 months. CONCLUSION: Despite the small sample size, the study showed a significant reduction in pad leakage and leaks per day over 24 h in the active treatment group over a 6-month period. Nearly half of patients on placebo had disabling urinary tract infection symptoms that required clinical override treatment.

Correlation between cystometric volumes, ATP release, and pH in women with overactive bladder versus controls.

AIMS: In the bladder, ATP is an important signaling molecule, which is released by bladder stretch and acid. We hypothesized that ATP might play a unique role in patients with OAB, characterized by low bladder volumes at first desire to void (FDV) and maximal cystometric capacity (MCC) and symptoms of frequency/urgency [mild bladder pain syndrome (BPS)]. Our aim was to investigate the correlation between ATP release and urodynamic parameters, as well as urine pH, in OAB patients. METHODS: Routine cystometry was performed in a consecutive series of 249 women. The voided urodynamic fluid (VUF) was stored at -20°C and ATP measured using bioluminescence. Catheter urine was collected for pH measurement. Correlations between two factors were tested by linear regression analysis. RESULTS: Subjects with urinary tract infection, voiding dysfunction, and detrusor overactivity (DO) were excluded. For OAB patients (n = 25), there was an inverse correlation between ATP concentration in VUF and FDV (r(2) = 0.25; P = 0.01) but not MCC. This was not seen in controls (n = 69). In OAB, but not controls, there was a significant reverse correlation (r(2) = 0.16; P = 0.047) between ATP in VUF and urine pH. Urine pH was not significantly correlated with MCC in either group. CONCLUSIONS: In OAB patients, ATP is an important factor for initial perception of need to urinate (as indicated by FDV). This is similar to our previous findings in patients with DO, suggesting that ATP may mediate initial afferent sensation in patients with bladder dysfunctions characterized by urgency. ATP release was also strongly affected by urine pH, in patients with OAB (at FDV).

Randomized controlled trial of cough test versus no cough test in the tension-free vaginal tape procedure: effect upon voiding dysfunction and 12-month efficacy.

INTRODUCTION AND HYPOTHESIS: This is a prospective randomized controlled trial of cough versus no cough test in the tension-free vaginal tape (TVT) procedure to determine its effect upon voiding dysfunction and 12-month efficacy. METHODS: The trial was conducted in a single tertiary urogynecology unit. Women ≥21 years old with primary urodynamic stress incontinence without voiding dysfunction were considered eligible. Participants were randomized to undergo the TVT procedure using either an intraoperative cough test or using no intraoperative cough test. Our hypothesis was that postoperative voiding dysfunction would be more common in the "no cough test" arm. The primary outcome was proportion of patients successfully completing a trial of void (TOV) within 24 h of catheter removal. Efficacy at 12 months comprised the secondary outcome. Participants were randomized using a computer-generated randomization sequence by an independent party who was not the operating surgeon. Due to the nature of the intervention to be tested, neither the patients nor the operating surgeons were blinded to the randomization process during the procedure. RESULTS: This trial is reported according to the recommendations of the 2010 CONSORT statement. In total, 94 women were recruited over a 4-year study period. Of these, 92 women were randomized (47 in the "cough" group and 45 in the "no cough" group). In one case, the TVT procedure was abandoned intraoperatively, leaving 91 women who underwent analysis. There was no significant difference in the proportion of women with a successful TOV within 24 h between the two arms (79% in the "cough" group versus 71% in the "no cough" group; p = 0.47). Efficacy data at 12 months were not significantly different between groups. CONCLUSION: Our data suggest that the performance of the intraoperative cough test during the TVT procedure does not reduce the incidence of postoperative voiding dysfunction (as determined by successful TOV within 24 h) nor affect efficacy. The removal of the cough test from the standard TVT technique may be appropriate.

Does monthly self-management of vaginal ring pessaries reduce the rate of adverse events? A clinical audit.

Objective: To determine the rate of adverse events (AE) in women who self-manage their vaginal ring pessary on a monthly basis. We hypothesised that the AE rate would be lower compared to previously published traditional management protocols. Study design: Audit study of 75 women with pelvic organ prolapse and/or stress incontinence, who were fitted with a vaginal ring pessary during a five-year period, and who have self-managed their vaginal rings for at least two years, in a tertiary referral urogynaecology clinic. Main outcome measures: AEs included vaginal bleeding, malodorous vaginal discharge, extrusion of the device, pain/discomfort, and disorders of defaecation or de novo urinary incontinence. AEs that led to discontinuation of usage were termed "major". Results: Of the 75 women who were taught to self-manage their ring pessary, 68 were initially successful. At a median follow-up of 50.5 months [IQR 43-76 months; median 4.2 years], 36 women (52.9%) were still using their ring pessary. Five women (7.4%) had vaginal erosions and bleeding leading them to cease pessary use (four proceeded to surgery). Three minor AEs were identified (4.4%), resolving after discontinuation of ring use two weeks. Thus, the overall AE rate was 11.8% (8/68). Conclusions: In contrast to previous published AE rates of 43-56% in women having ring changes at a clinic every 4-6 months, the AE rate was 12% in the women who performed monthly self-management of vaginal ring pessaries. Such information should be made available to patients considering a vaginal ring pessary.

Does adenosine triphosphate released into voided urodynamic fluid contribute to urgency signaling in women with bladder dysfunction?

PURPOSE: Adenosine triphosphate released from urothelium during stretch stimulates afferent nerves and conveys information on bladder fullness. We measured adenosine triphosphate released during cystometric bladder filling in women with idiopathic detrusor overactivity and stress incontinence (controls), and assessed whether the level of released adenosine triphosphate is related to cystometric parameters. MATERIALS AND METHODS: Routine cystometry was done in 51 controls and 48 women with detrusor overactivity who were 28 to 87 years old. Voided urodynamic fluid was collected and stored at -30 C. Adenosine triphosphate was measured by a bioluminescence assay. RESULTS: Adenosine triphosphate levels were similar in voided urodynamic fluid of controls and patients with detrusor overactivity (p = 0.79). A significant inverse correlation was seen between adenosine triphosphate and maximal cystometric capacity in controls (p = 0.013), and between voided volume and adenosine triphosphate in controls (p = 0.015) and detrusor overactivity cases (p = 0.019). A significant correlation between first desire to void and adenosine triphosphate was also noted in detrusor overactivity cases (p = 0.033) but not in controls (p = 0.58). No correlation was seen between adenosine triphosphate and detrusor pressure during filling or voiding. CONCLUSIONS: Adenosine triphosphate measurement in voided urodynamic fluid is a novel approach to understanding signals that may contribute to the urgency sensation (a sudden compelling desire to pass urine). The inverse correlation between adenosine triphosphate in voided urodynamic fluid and first desire to void suggests that adenosine triphosphate has a role in modulating the early filling sensation in patients with detrusor overactivity.

The colorectal cancer disease-specific transcriptome may facilitate the discovery of more biologically and clinically relevant information.

BACKGROUND: To date, there are no clinically reliable predictive markers of response to the current treatment regimens for advanced colorectal cancer. The aim of the current study was to compare and assess the power of transcriptional profiling using a generic microarray and a disease-specific transcriptome-based microarray. We also examined the biological and clinical relevance of the disease-specific transcriptome. METHODS: DNA microarray profiling was carried out on isogenic sensitive and 5-FU-resistant HCT116 colorectal cancer cell lines using the Affymetrix HG-U133 Plus2.0 array and the Almac Diagnostics Colorectal cancer disease specific Research tool. In addition, DNA microarray profiling was also carried out on pre-treatment metastatic colorectal cancer biopsies using the colorectal cancer disease specific Research tool. The two microarray platforms were compared based on detection of probesets and biological information. RESULTS: The results demonstrated that the disease-specific transcriptome-based microarray was able to out-perform the generic genomic-based microarray on a number of levels including detection of transcripts and pathway analysis. In addition, the disease-specific microarray contains a high percentage of antisense transcripts and further analysis demonstrated that a number of these exist in sense:antisense pairs. Comparison between cell line models and metastatic CRC patient biopsies further demonstrated that a number of the identified sense:antisense pairs were also detected in CRC patient biopsies, suggesting potential clinical relevance. CONCLUSIONS: Analysis from our in vitro and clinical experiments has demonstrated that many transcripts exist in sense:antisense pairs including IGF2BP2, which may have a direct regulatory function in the context of colorectal cancer. While the functional relevance of the antisense transcripts has been established by many studies, their functional role is currently unclear; however, the numbers that have been detected by the disease-specific microarray would suggest that they may be important regulatory transcripts. This study has demonstrated the power of a disease-specific transcriptome-based approach and highlighted the potential novel biologically and clinically relevant information that is gained when using such a methodology.

RALB GTPase: a critical regulator of DR5 expression and TRAIL sensitivity in KRAS mutant colorectal cancer.

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.

Clinical determinants of response to irinotecan-based therapy derived from cell line models.

PURPOSE: In an attempt to identify genes that are involved in resistance to SN38, the active metabolite of irinotecan (also known as CPT-11), we carried out DNA microarray profiling of matched HCT116 human colon cancer parental cell lines and SN38-resistant cell lines following treatment with SN38 over time. EXPERIMENTAL DESIGN: Data analysis identified a list of genes that were acutely altered in the parental cells following SN38 treatment as well as constitutively altered in the SN38-resistant cells. RESULTS: Independent validation of 20% of these genes by quantitative reverse transcription-PCR revealed a strong correlation with the microarray results: Pearson's correlation was 0.781 (r(2) = 0.61, P < 0.000001) for those genes that were acutely altered in the parental setting following SN38 treatment and 0.795 (r(2) = 0.63, P < 0.000002) for those genes that were constitutively altered in the SN38-resistant cells. We then assessed the ability of our in vitro-derived gene list to predict clinical response to 5-fluorouracil/irinotecan using pretreatment metastatic biopsies from responding and nonresponding colorectal cancer patients using both unsupervised and supervised approaches. When principal components analysis was used with our in vitro classifier gene list, a good separation between responding and nonresponding patients was obtained, with only one nonresponding and two responding patients separating with the incorrect groups. Supervised class prediction using support vector machines algorithm identified a 16-gene classifier with 75% overall accuracy, 81.8% sensitivity, and 66.6% specificity. CONCLUSIONS: These results suggest that in vitro-derived gene lists can be used to predict clinical response to chemotherapy in colorectal cancer.

Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer.

DNA microarray analysis was used to analyze the transcriptional profile of HCT116 colorectal cancer cells that were treated with 5-fluorouracil (5-FU) or oxaliplatin and selected for resistance to these agents. Bioinformatic analyses identified sets of genes that were constitutively dysregulated in drug-resistant cells and transiently altered following acute exposure of parental cells to drug. We propose that these genes may represent molecular signatures of sensitivity to 5-FU and oxaliplatin. Using real-time reverse transcription-PCR (RT-PCR), the robustness of our microarray data was shown with a strong overall concordance of expression trends for > or =82% (oxaliplatin) and > or =85% (5-FU) of a representative subset of genes. Furthermore, strong correlations between the microarray and real-time RT-PCR measurements of average fold changes in gene expression were observed for both the 5-FU (R(2) > or = 0.73) and oxaliplatin gene sets (R(2) > or = 0.63). Functional analysis of three genes identified in the microarray study [prostate-derived factor (PDF), calretinin, and spermidine/spermine N(1)-acetyl transferase (SSAT)] revealed their importance as novel regulators of cytotoxic drug response. These data show the power of this novel microarray-based approach to identify genes which may be important markers of response to treatment and/or targets for therapeutic intervention.

The role of spermidine/spermine N1-acetyltransferase in determining response to chemotherapeutic agents in colorectal cancer cells.

Polyamines have been shown to play a role in the growth and survival of several solid tumors, including colorectal cancer. We identified the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as being one of the most highly inducible genes in two DNA microarray screens to identify novel determinants of response to chemotherapeutic agents in colorectal cancer. SSAT was shown to be inducible in response to 5-fluorouracil (5-FU) or oxaliplatin in parental and drug-resistant HCT116 cell lines. It was also shown that SSAT mRNA was up-regulated in response to 5-FU or oxaliplatin in a panel of six colorectal cancer cell lines. The polyamine analogue N(1),N(11)-diethylnorspermine (DENSpm) depletes polyamine pools and potently induces SSAT. We evaluated the effect of combining DENSpm with chemotherapeutic agents in HCT116 p53(+/+) cells and in HCT116 drug-resistant daughter cell lines. Western blot analyses showed that SSAT protein expression was dramatically enhanced when DENSpm was combined with oxaliplatin or 5-FU in HCT116 p53(+/+) cells. Using cell viability assays and flow cytometry, synergistic induction of cell death was observed following cotreatment of HCT116 p53(+/+) cells with DENSpm and each chemotherapeutic agent. Of note, this combined therapy increased the chemosensitivity of cells rendered resistant to each of these chemotherapeutic agents. Small interfering RNA-mediated down-regulation of SSAT resulted in loss of synergy between DENSpm and these agents. These results show that SSAT plays an important role in regulating cell death following combined cytotoxic drug and DENSpm treatment. Furthermore, DENSpm sensitizes both sensitive and resistant cells to chemotherapeutic agents. Taken together, these results suggest that SSAT may be an important target for therapeutic intervention in colorectal cancer.

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells.

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that IFN-gamma and doxorubicin cotreatment synergistically induced apoptosis in MDA435 breast cancer cells in a signal transducer and activator of transcription 1-dependent manner. In this study, we found that this synergy was caspase-8 dependent. In addition, we found that IFN-gamma down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-gamma down-regulated c-FLIP in a manner that was dependent on the transcription factors signal transducer and activator of transcription 1 and IFN regulatory factor-1. However, IFN-gamma had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-gamma treatment. Characterization of the functional significance of c-FLIP modulation by small interfering RNA gene silencing and stable overexpression studies revealed it to be a key regulator of IFN-gamma- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-gamma-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitized MDA435 cells to other chemotherapies, including etoposide, mitoxantrone, and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

The Unfolded Protein Response: A Novel Therapeutic Target for Poor Prognostic BRAF Mutant Colorectal Cancer.

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

Drug resistance, predictive markers and pharmacogenomics in colorectal cancer.

Resistance to chemotherapy limits the effectiveness of current cancer therapies, including those used to treat colorectal cancer, which is the second most common cause of cancer death in Europe and the United States. 5-Fluorouracil-based chemotherapy regimens are the standard treatment for colorectal cancer in both the adjuvant and advanced disease settings. Drug resistance is thought to cause treatment failure in over 90% of patients with metastatic cancer, while drug resistant micrometastic tumour cells may also reduce the impact of adjuvant chemotherapy treatment. The identification of panels of biomarkers that not only identify those patients most likely to benefit from chemotherapy treatment, but also which chemotherapies to use, would be a major advance. In this review, we describe molecular mechanisms of drug resistance that may be relevant to colorectal cancer. We also describe the results of predictive biomarker studies in this disease. Finally, we discuss how pharmacogenomics and other high through-put technologies may impact on the clinical management of colorectal cancer in the future.

Predicting the outcome of chemotherapy for colorectal cancer.

Colorectal cancer is the second leading cause of cancer-related deaths in the Western world. Recently, improvements have been made in treating patients with advanced colorectal cancer; however, response rates still remain low at only 40-50% following combination therapy. The major limitation in treating these patients is the development of drug resistance. Therefore, there is a need to identify which patients will respond to a given chemotherapy regime so that they will be spared the unnecessary time and toxicity of being placed on a regime from which they will derive no benefit. It is also widely accepted that exposure to these chemotherapies themselves can induced acute resistance. Recent developments have been made in predicting response to chemotherapy using global approaches, with the ultimate aim of individualising patient treatment and improving overall survival rates.

Effect of p53 status and STAT1 on chemotherapy-induced, Fas-mediated apoptosis in colorectal cancer.

We investigated the role of p53 and the signal transducer and activator of transcription 1 (STAT1) in regulating Fas-mediated apoptosis in response to chemotherapies used to treat colorectal cancer. We found that 5-fluorouracil (5-FU) and oxaliplatin only sensitized p53 wild-type (WT) colorectal cancer cell lines to Fas-mediated apoptosis. In contrast, irinotecan (CPT-11) and tomudex sensitized p53 WT, mutant, and null cells to Fas-mediated cell death. Furthermore, CPT-11 and tomudex, but not 5-FU or oxaliplatin, up-regulated Fas cell surface expression in a p53-independent manner. In addition, increased Fas cell surface expression in p53 mutant and null cell lines in response to CPT-11 and tomudex was accompanied by only a slight increase in total Fas mRNA and protein expression, suggesting that these agents trigger p53-independent trafficking of Fas to the plasma membrane. Treatment with CPT-11 or tomudex induced STAT1 phosphorylation (Ser727) in the p53-null HCT116 cell line but not the p53 WT cell line. Furthermore, STAT1-targeted small interfering RNA (siRNA) inhibited up-regulation of Fas cell surface expression in response to CPT-11 and tomudex in these cells. However, we found no evidence of altered Fas gene expression following siRNA-mediated down-regulation of STAT1 in drug-treated cells. This suggests that STAT1 regulates expression of gene(s) involved in cell surface trafficking of Fas in response to CPT-11 or tomudex. We conclude that CPT-11 and tomudex may be more effective than 5-FU and oxaliplatin in the treatment of p53 mutant colorectal cancer tumors by sensitizing them to Fas-mediated apoptosis in a STAT1-dependent manner.

Role of genomic markers in colorectal cancer treatment.

For the last four decades, fluorouracil (FU) has been the main treatment of choice in colorectal cancer (CRC) in both the advanced and adjuvant settings. In the advanced setting, FU monotherapy produces response rates of only 10% to 20%. Furthermore, in resected stage III CRC, FU monotherapy has increased overall survival by only 20%. The combination of FU with newer therapies such as oxaliplatin and irinotecan has significantly improved response rates to 40% to 50%. Despite these improvements, more than half of advanced CRC patients derive no benefit from treatment; this is due to either acquired or inherent drug resistance. This review aims to highlight the current prognostic and predictive markers that have been identified for CRC to date. The limited use of these predictive markers underscores the importance of and need for multiple marker testing in order to improve response rates and decrease toxicity. This review will also focus on high throughput methods to identify panels of predictive markers for CRC, which ultimately aim to tailor treatment according to an individual patient and tumor profile.

The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.