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PDF file - 261K, List showing 1986 genes that are uniquely altered following SN38 treatment over time in HCT116 p53null cells only. Genes have been normalised and filtered as stated in the materials and methods

Caspase-8 dependent apoptosis induced by IR

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.

Table S1: Top 20 pathways identified by IPA containing genes significantly upregulated between poor and good prognostic BRAFMT stage II/III CRC in GSE39582; Table S2: Top 20 pathways identified by IPA containing genes significantly downregulated between poor and good prognostic BRAFMT stage II/III CRC in GSE39582; Table S3: Top 20 pathways identified by IPA containing genes differentially expressed between poor (disease free survival 5 years) in E-MTAB-863 and E-MTAB-864; Table S4: Results of siRNA screen in BRAFMT CRC.

PDF file - 321K, List showing 2544 genes that are uniquely altered following SN38 treatment over time in HCT116 p53wild-type cells only. Genes have been normalised and filtered as stated in the materials and methods

Supplementary figure 1: A +B. Hierarchical clustering of microdissected stromal and epithelial (A) and FACS sorted endothelial, fibroblast, epithelial and leukocyte (B) cell populations using previously published non-epithelial derived gene list confirms purity of cell populations. C and D. PCA plot of data presented in top panels highlights clear stratification of samples based on cell lineage using this gene signature. E. Expression values for IFNG expression according to cell-of-origin confirms immune-derived nature of signal. Supplementary figure 2: Association of high PD-L1 expression with MSI subtype and high immune infiltrate. Supplementary figure 3: A. Dot and boxplots of PD-L1 mean gene expression values stratified by MSI status highlights significant association between PD-L1 transcription levels and MSI genotype. B. (Left) Hierarchical clustering of stage II/III CRC cohort based on expression profiles of CD274, CTLA4, LAG3 and IDO1. (Right) Significant upregulation of CD274, IDO1, CTLA4 and LAG3 genes in the PD-L1high subgroup. C. (Left) Dot plot and boxplot of IFNG expression levels stratified by PD-L1-subgroup. (Right) Significant upregulation of IFNG gene in the PD-L1high subgroup.

Supplementary materials and methods

PDF file - 41K, QPCR validation.

PDF file - 61K, unsupervised top 10 positive and negative correlated genes - 5-FU basal.

IR-induced changes in cell surface expression of death receptors

PDF file - 62K, unsupervised top 10 positive and negative correlated genes - clinical.

Response of H460 and A549 cells to IR

PDF file - 132K, List showing 752 genes that are commonly altered following SN38 treatment over time in isogenic HCT116 p53wild-type and HCT116 p53null cells. Genes have been normalised and filtered as stated in the materials and methods

PDF file - 64K, unsupervised top 10 positive and negative correlated genes - SN38 inducible.

PDF file - 32K, QPCR validations.

PDF file - 35K, Real-time PCR validation of HCT116 p53 null cells following treatment with 5nM SN38 for 6, 12 and 24h. This table shows the Pearson's correlation and p values for each individual gene

PDF file - 11K, Results from KEGG pathway analysis for p53 wild-type and p53 null basal comparison. The table shows the number of genes in each pathway and the p value associated with that pathway calculated using hypergeometric statistics. The pathways that are highlighted in bold are those that are statistically significant (p

PDF file - 62K, unsupervised top 10 positive and negative correlated genes - 5-FU inducible.

PDF file - 214K, List showing 2102 genes that are constitutively altered between isogenic HCT116 p53wild-type and HCT116 p53null cells. Genes have been normalised and filtered as stated in the materials and methods

Combination of SAHA with IR in H460 and A549 cells using different treatment schedules

PDF file - 11K, Real-time PCR validation of genes basally altered between HCT116 p53 null cells and HCT116 p53 wild-type cells. This table shows the p53 null fold change by microarray and the p53 null fold change by real-time PCR, all compared to the p53wild-type cell expression values

PDF file - 63K, unsupervised top 10 positive and negative correlated genes - SN38 basal.

A recent phase II study of patients with metastatic colorectal carcinoma showed that mismatch repair gene status was predictive of clinical response to PD-1–targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV colorectal carcinoma, suggesting that the amount of PD-L1 protein expression could identify late-stage patients who might benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic colorectal carcinoma are also present in stage II/III colorectal carcinoma, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a statistically significant association of PD-L1 gene expression with MSI in early-stage colorectal carcinoma (P < 0.001) and show that, unlike in non–colorectal carcinoma tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1low vs. PD-L1high HR = 9.09; CI, 2.11–39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI, 1.10–22.35). Building on the promising results from the metastatic colorectal carcinoma PD-1–targeting trial, we provide compelling evidence that patients with PD-L1high/MSI/immunehigh stage II/III colorectal carcinoma should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment. Cancer Immunol Res; 4(7); 582–91. ©2016 AACR.

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8. Mol Cancer Ther; 11(1); 119–31. ©2011 AACR.

Supplementary Figure S7. MEK1/2 regulates levels of decoy MET in BRAFMT in vitro and in vivo models.

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools.Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer (n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136).Results: We identified 3 prognostic biomarkers (P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer.The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients (P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype.Conclusions: The integration of a systems-biology–based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200–12. ©2016 AACR.

Purpose:The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer.Experimental Design:Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial (n = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial (n = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer.Results:Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer.Conclusions:DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.

This Supplementary Figure indicates the project plan

Clinical relevance of EphA2 expression in CRC tissues.

PDF file - 1.8K, Percentage of sub-G1 phase apoptotic cells and apoptotic pathway induced by Ran silencing in different cell lines. (A) Cells were harvested 72 hours post-infection, fixed and stained with Propidium iodide. Significantly fewer Sub-G1 phase apoptotic cells were observed in the immortalized human embryonic kidney 293 (HEK 293) cell line compared to cancer cell lines from breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (HCT116 and DLD-1) upon Ran silencing (p < 0.01). (B) Western blots of apoptosis mediators in immortalized HEK293, breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (DLD-1 and HCT116) cancer cell lines at 72 hours post-infection with Scr or Ran shRNAs.

Supplementary Data from Prostate-derived factor—a novel inhibitor of drug-induced cell death in colon cancer cells

Kaplan-Meier estimates for disease-free and overall survival for n=120 stage III patients of the discovery cohort categorized based on TN staging, tumor location and lymphovascular invasion. Additional exploratory analyses investigated the prognostic value of the APOPTO-CELL-PC3 signature within each sub-group identified by the clinical features.

Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.Experimental Design: Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers.Results: Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways.Conclusion: Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. Clin Cancer Res; 18(2); 380–91. ©2011 AACR.

Supplementary Table 4: Differentially expressed gene list contrasting tumour region of origin between Lymph Node and Invasive Front.

Supplementary Results

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

PDF file - 235K, Clinical relevance of AXL in CRC tissues.

PDF file - 223K, Univariate and multivariate analyses for OS, DSS, DFS and AXL-LOW and AXL-HIGH in stage II/III CRC group (publicly-available CRC microarray dataset).

Purpose: A major factor limiting the effective clinical management of colorectal cancer (CRC) is resistance to chemotherapy. Therefore, the identification of novel, therapeutically targetable mediators of resistance is vital.Experimental design: We used a CRC disease-focused microarray platform to transcriptionally profile chemotherapy-responsive and nonresponsive pretreatment metastatic CRC liver biopsies and in vitro samples, both sensitive and resistant to clinically relevant chemotherapeutic drugs (5-FU and oxaliplatin). Pathway and gene set enrichment analyses identified candidate genes within key pathways mediating drug resistance. Functional RNAi screening identified regulators of drug resistance.Results: Mitogen-activated protein kinase signaling, focal adhesion, cell cycle, insulin signaling, and apoptosis were identified as key pathways involved in mediating drug resistance. The G-protein–coupled receptor galanin receptor 1 (GalR1) was identified as a novel regulator of drug resistance. Notably, silencing either GalR1 or its ligand galanin induced apoptosis in drug-sensitive and resistant cell lines and synergistically enhanced the effects of chemotherapy. Mechanistically, GalR1/galanin silencing resulted in downregulation of the endogenous caspase-8 inhibitor FLIPL, resulting in induction of caspase-8–dependent apoptosis. Galanin mRNA was found to be overexpressed in colorectal tumors, and importantly, high galanin expression correlated with poor disease-free survival of patients with early-stage CRC.Conclusion: This study shows the power of systems biology approaches to identify key pathways and genes that are functionally involved in mediating chemotherapy resistance. Moreover, we have identified a novel role for the GalR1/galanin receptor–ligand axis in chemoresistance, providing evidence to support its further evaluation as a potential therapeutic target and biomarker in CRC. Clin Cancer Res; 18(19); 5412–26. ©2012 AACR.

Unadjusted and multivariate Cox proportional hazards analyses to examine the association of APOPTO-CELL and APOPTO-CELL-PC3 signatures with DFS in n=157 stage III patients of the expansion cohort.

PDF file - 179K, Effect of AXL siRNA on migration, proliferation and survival of CRC cells.

Supplementary Figures S1-S2 and Supplementary Tables S1-S3.

PDF file - 3.6K, Cancer cells with K-Ras activation mutation (HCT116/Hkh-2 isogenic pair) are more susceptible to Ran silencing-induced apoptosis.

PDF file - 94K, Sensitivity of parental and invasive CRC cells to chemotherapy and EGFR targeted therapies.

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 (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.

CCR Translation for this Article from Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

PDF file - 544K, In vitro and clinical microarray genelists

PDF file - 355K, supplementary figures S1. RNAi screen S2. QPCR S3. Cell viability analysis S4. Apoptosis analysis S5. FLIPL