Vicky M. Coyle

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]

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: Pearsons correlation was 0.781 (r2 = 0.61, P < 0.000001) for those genes that were acutely altered in the parental setting following SN38 treatment and 0.795 (r2 = 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.

Molecular Subtypes and Personalized Therapy in Metastatic Colorectal Cancer

Development of colorectal cancer occurs via a number of key pathways, with the clinicopathological features of specific subgroups being driven by underlying molecular changes. Mutations in key genes within the network of signalling pathways have been identified; however, therapeutic strategies to target these aberrations remain limited. As understanding of the biology of colorectal cancer has improved, this has led to a move toward broader genomic testing, collaborative research and innovative, adaptive clinical trial design. Recent developments in therapy include the routine adoption of wider mutational spectrum testing prior to use of targeted therapies and the first promise of effective immunotherapy for colorectal cancer patients. This review details current biomarkers in colorectal cancer for molecular stratification and for treatment allocation purposes, including open and planned precision medicine trials. Advances in our understanding, therapeutic strategy and technology will also be outlined.

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug. Mol Cancer Ther; 11(8); 1724–34. ©2012 AACR.

Natural killer-like signature observed post therapy in locally advanced rectal cancer is a determinant of pathological response and improved survival

Around 12–15% of patients with locally advanced rectal cancer undergo a pathologically complete response (tumor regression grade 4) to long-course preoperative chemoradiotherapy; the remainder exhibit a spectrum of tumor regression (tumor regression grade 1–3). Understanding therapy-related transcriptional alterations may enable better prediction of response as measured by progression-free and overall survival, in addition to aiding the development of improved strategies based on the underlying biology of the disease. To this end, we performed high-throughput gene expression profiling in 40 pairs of formalin-fixed paraffin-embedded rectal cancer biopsies and matched resections following long-course preoperative chemoradiotherapy (discovery cohort). Differential gene expression analysis was performed contrasting tumor regression grades in resections. Enumeration of the tumor microenvironment cell population was undertaken using in silico analysis of the transcriptional data, and real-time PCR validation of NCR1 undertaken. Immunohistochemistry and survival analysis was used to measure CD56+ cell populations in an independent cohort (n=150). Gene expression traits observed following long-course preoperative chemoradiotherapy in the discovery cohort suggested an increased abundance of natural killer cells in tumors that displayed a clinical response to CRT in a tumor regression grade-dependent manner. CD56+ natural killer-cell populations were measured by immunohistochemistry and found to be significantly higher in tumor regression grade 3 patients compared with tumor regression grade 1–2 in the validation cohort. Furthermore, it was observed that patients positive for CD56 cells after therapy had a better overall survival (HR=0.282, 95% CI=0.109–0.729, χ2=7.854, P=0.005). In conclusion, we have identified a novel post-therapeutic natural killer-like transcription signature in patients responding to long-course preoperative chemoradiotherapy. Furthermore, patients with a higher abundance of CD56-positive natural killer cells post long-course preoperative chemoradiotherapy had better overall survival. Therefore, harnessing a natural killer-like response after therapy may improve outcomes for locally advanced rectal cancer patients. Finally, we hypothesize that future assessment of this natural killer-like response in on-treatment biopsy material may inform clinical decision-making for treatment duration.

Transcriptional Subtyping and CD8 Immunohistochemistry Identifies Patients With Stage II and III Colorectal Cancer With Poor Prognosis Who Benefit From Adjuvant Chemotherapy

Purpose Transcriptomic profiling of colorectal cancer (CRC) has led to the identification of four consensus molecular subtypes (CMS1 to 4) that have prognostic value in stage II and III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors; however, the clinical value of these classification systems in the prediction of response to standard-of-care adjuvant chemotherapy remains unknown. Patients and Methods Using samples from four European sites, we assembled a novel cohort of patients with stage II and III CRC (n = 156 samples) and performed transcriptomic profiling and targeted sequencing and generated a tissue microarray to enable integrated multiomics analyses. We also accessed data from two published cohorts of patients with stage II and III CRC: GSE39582 and GSE14333 (n = 479 and n = 185 samples, respectively). Results The epithelial-rich CMS2 subtype of CRC benefitted significantly from treatment with adjuvant chemotherapy in both stage II and III disease (P = .02 and P < .001, respectively), whereas the CMS3 subtype significantly benefitted in stage III only (P = .001). After CRIS substratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from treatment with adjuvant chemotherapy in stage II and III disease (P = .0081 and P < .001, respectively), whereas the CRIS-D subtype significantly benefitted in stage III only (P = .0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk for relapse in both stage II and III disease (log-rank P = .0031; hazard ratio, 12.18 [95% CI, 1.51 to 98.58]). Conclusion Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying patients with poor prognostic stage II and III disease who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for patients with stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

A first-in-human phase I study to determine the maximum tolerated dose of the oral Src/ABL inhibitor AZD0424

Background:Src is involved in cancer invasion and metastasis. AZD0424, an oral inhibitor of Src and ABL1, has shown evidence of anti-tumour activity in pre-clinical studies.Methods:A phase Ia, dose escalation study was performed to assess the safety of continuous oral dosing with AZD0424 in advanced solid tumours. Secondary objectives included investigation of AZD0424 pharmacokinetics, effect on Src activity using markers of bone turnover, and anti-tumour activity.Results:41 patients were treated; 34 received AZD0424 once-daily at doses ranging from 5 mg to 150 mg, and 7 received 40 mg bi-daily 41.5% of patients experienced at least one AZD0424-related adverse event that was Grade 3–5 in severity, with patients treated at doses above 60 mg per day experiencing multiple treatment-related toxicities. The most commonly observed AZD0424-related adverse events were nausea, fatigue, anorexia and alopecia. Cmax and AUC increased linearly with dose and the mean±standard deviation t1/2 was 8.4±2.8 h. Clear evidence of Src target inhibition was seen at doses ⩾20 mg per day. No responses were observed and 7 patients (17.1%) achieved stable disease lasting 6 weeks or more.Conclusions:AZD0424 displayed no evidence of efficacy as monotherapy despite a clear pharmacodynamic effect. Further evaluation of AZD0424 monotherapy in patients with solid tumours is not recommended.

Inhibition of FGFR4 increases oxaliplatin and 5-fluorouracil sensitivity in Kras wild-type and mutant colorectal cancer cells.

e14087 Background: The discovery of underlying mechanisms of drug resistance and the development of novel agents to target these pathways is a priority for patients with advanced colorectal cancer (CRC). The fibroblast growth factor receptor 4 (FGFR4) is frequently overexpressed in solid tumours, such as CRC, and has been shown to be an important regulator of cancer cell growth and motility. The aim of this study was to identify novel targets whose knock-down is important in mediating sensitivity to 5-FU and oxaliplatin in Kras wild-type (WT) and mutant (MT) CRC models. METHODS Transcriptional profiling (Almac Diagnostics CRC Disease Specific Array) of pre-treatment metastatic CRC liver biopsies and oxaliplatin/5-FU resistant HCT116 cell lines followed by Metacoreä and Gene Set Enrichment Analysis (GSEA) were used to identify individual genes from novel drug-sensitivity pathways for incorporation into a RNAi screen. RESULTS We identified panels of in vitro and clinical genes whose expression is altered (acutely and basally) between sensitive and 5-FU- or oxaliplatin-resistant models. The significant pathways involved in 5-FU/oxaliplatin resistance included cell cycle, focal adhesion, insulin and MAPK signalling. In the MAPK pathway, we found that FGFR4 silencing potently increased apoptosis in KrasWT and MT CRC cells, and this was further enhanced when FGFR4 siRNA was combined with 5-FU or oxaliplatin. Interestingly, FGFR4 inhibition completely inhibited migration of KrasMT HCT116 cells. Mechanistically, we found that FGFR4, silencing resulted in strong inhibition of STAT3 activity in both KrasWT and MT CRC cells. CONCLUSIONS This study demonstrates the utility of microarray expression data, obtained from pre-clinical and clinical samples, and analyzed by pathway and gene set enrichment analysis to identify pathways of oxaliplatin/5-FU sensitivity in CRC. In addition FGFR4 inhibition in combination with 5-FU or oxaliplatin could represent a novel treatment strategy for KrasMT and WT CRC tumours. We are currently investigating FGFR4 small molecule inhibitors in preclinical in vitro and in vivo models.