Wendy L. Allen

c-FLIP inhibits chemotherapy-induced colorectal cancer cell death

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIPL and c-FLIPS with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53+/+, RKO), null (HCT116p53−/−) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIPL, but not c-FLIPS, potently inhibited apoptosis induced by chemotherapy in HCT116p53+/+ cells, suggesting that c-FLIPL was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIPL synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAIL-independent, DR5- and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

The interaction of thymidylate synthase expression with p53-regulated signaling pathways in tumor cells.

Thymidylate synthase (TS) is a chemotherapeutic target for the fluoropyrimidine 5-fluorouracil (5-FU) and antifolate tomudex (TDX). Using the MCF-7 breast cancer line, we have developed a cell line with inducible TS expression termed M7TS90. Inducible TS expression in this line resulted in a moderate (approximately 3-fold) increase in 5-FU 50% inhibitory concentration at 72 hours (IC-50(72 h)) dose and a dramatic (approximately 24-fold) increase in the IC-50(72 h) dose of TDX, but did not affect chemosensitivity to cisplatin, oxaliplatin, irinotecan, and paclitaxel. In the absence of drug treatment, inducible TS expression had no effect on expression of the p53 tumor suppressor gene. However, TS induction abrogated p53, p21, Fas, and Bak induction in response to TDX, but not 5-FU. Similarly, downregulation of Bcl-2 was reversed by inducible TS expression in TDX, but not 5-FU-treated cells. Our results indicate that inducible TS expression in M7TS90 cells modulates p53 and p53 target gene expression in response to TDX, but not 5-FU.

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.

Implementing prognostic and predictive biomarkers in CRC clinical trials.

Over the past two decades, several protein and genomic markers have refined the prognostic information of colorectal cancer (CRC) and helped to predict which patient group may benefit most from systemic treatment or targeted therapies. Of all these markers, KRAS represents the first biomarker integrated into clinical practice for CRC. Microarray-based gene-expression profiling has been used to identify prognostic signatures and to a lesser degree predictive signatures in CRC; however, common challenges with these types of studies are clinical study design, reproducibility, interpretation and reporting of the results. We focus on the clinical application of a range of published prognostic and predictive protein and genomic markers in CRC and discuss the different challenges associated with microarray-based gene-expression profiling. While none of these genomic signatures is currently in routine clinical use in CRC, novel adaptive clinical trial designs that incorporate putative genomic prognostic/predictive markers in prospective randomized trials, will enable a clinical validation of these markers and may facilitate the implementation of these biomarkers into routine medical practice.

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.

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.

The Roles of Thymidylate Synthase and p53 in Regulating Fas- Mediated Apoptosis in Response to Antimetabolites

Fas (CD95/Apo-1) is a member of the tumor necrosis factor receptor family. Receptor binding results in activation of caspase 8, leading to activation of proapoptotic downstream molecules. We found that expression of Fas was up-regulated >10-fold in MCF-7 breast and HCT116 and RKO colon cancer cell lines after treatment with IC60 doses of 5-fluorouracil (5-FU) and raltitrexed (RTX). Combined treatment with the agonistic Fas antibody CH-11 and either 5-FU or RTX resulted in a highly synergistic induction of apoptosis in these cell lines. Similar results were obtained for another antifolate, Alimta. Induction of thymidylate synthase expression inhibited Fas induction in response to RTX and Alimta, but not in response to 5-FU. Furthermore, thymidylate synthase induction abrogated the synergy between CH-11 and both antifolates but had no effect on the synergistic interaction between 5-FU and CH-11. Inactivation of p53 in MCF-7 and HCT116 cell lines blocked 5-FU- and antifolate-mediated up-regulation of Fas. Furthermore, Fas was not up-regulated in response to 5-FU or antifolates in the p53-mutant H630 colon cancer cell line. Lack of Fas up-regulation in the p53-null and -mutant lines abolished the synergistic interaction between 5-FU and CH-11. Interestingly, synergy was still observed between the antifolates and CH-11 in the p53-null HCT116 and p53-mutant H630 cell lines, although this was significantly reduced compared with the p53 wild-type cell lines. Our results indicate that Fas is an important mediator of apoptosis in response to both 5-FU and antifolates.

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 N1-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 N1,N11-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. [Mol Cancer Ther 2007;6(1):128–37]

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-γ 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-γ down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-γ 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-γ had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-γ 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-γ– 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-γ–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. [Mol Cancer Ther 2007;6(5):1544–51]

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286–9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial–mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095–104. ©2016 AACR. See related commentary by Morris and Kopetz, p. 3989