The ZEB2-dependent EMT transcriptional programme drives therapy resistance by activating nucleotide excision repair genes ERCC1 and ERCC4 in colorectal cancer.

Resistance to adjuvant chemotherapy is a major clinical problem in the treatment of colorectal cancer (CRC). The aim of this study was to elucidate the role of an epithelial to mesenchymal transition (EMT)-inducing protein, ZEB2, in chemoresistance of CRC, and to uncover the underlying mechanism. We performed IHC for ZEB2 and association analyses with clinical outcomes on primary CRC and matched CRC liver metastases in compliance with observational biomarker study guidelines. ZEB2 expression in primary tumours was an independent prognostic marker of reduced overall survival and disease-free survival in patients who received adjuvant FOLFOX chemotherapy. ZEB2 expression was retained in 96% of liver metastases. The ZEB2-dependent EMT transcriptional programme activated nucleotide excision repair (NER) pathway largely via upregulation of the ERCC1 gene and other components in NER pathway, leading to enhanced viability of CRC cells upon oxaliplatin treatment. ERCC1-overexpressing CRC cells did not respond to oxaliplatin in vivo, as assessed using a murine orthotopic model in a randomised and blinded preclinical study. Our findings show that ZEB2 is a biomarker of tumour response to chemotherapy and risk of recurrence in CRC patients. We propose that the ZEB2-ERCC1 axis is a key determinant of chemoresistance in CRC.

Intratumoural immune signature to identify patients with primary colorectal cancer who do not require follow-up after resection: an observational study.

BACKGROUND: Following surgical and adjuvant treatment of primary colorectal cancer, many patients are routinely followed up with axial imaging (most commonly computerised tomography imaging) and blood carcinoembryonic antigen (a tumour marker) testing. Because fewer than one-fifth of patients will relapse, a large number of patients are followed up unnecessarily. OBJECTIVES: To determine whether or not the intratumoural immune signature could identify a cohort of patients with a relapse rate so low that follow-up is unnecessary. DESIGN: An observational study based on a secondary tissue collection of the tumours from participants in the FACS (Follow-up After Colorectal Cancer Surgery) trial. SETTING AND PARTICIPANTS: Formalin-fixed paraffin-embedded tumour tissue was obtained from 550 out of 1202 participants in the FACS trial. Tissue microarrays were constructed and stained for cluster of differentiation (CD)3+ and CD45RO+ T lymphocytes as well as standard haematoxylin and eosin staining, with a view to manual and, subsequently, automated cell counting. RESULTS: The tissue microarrays were satisfactorily stained for the two immune markers. Manual cell counting proved possible on the arrays, but manually counting the number of cores for the entire study was found to not be feasible; therefore, an attempt was made to use automatic cell counting. Although it is clear that this approach is workable, there were both hardware and software problems; therefore, reliable data could not be obtained within the time frame of the study. LIMITATIONS: The main limitations were the inability to use machine counting because of problems with both hardware and software, and the loss of critical scientific staff. Findings from this research indicate that this approach will be able to count intratumoural immune cells in the long term, but whether or not the original aim of the project proved possible is not known. CONCLUSIONS: The project was not successful in its aim because of the failure to achieve a reliable counting system. FUTURE WORK: Further work is needed to perfect immune cell machine counting and then complete the objectives of this study that are still relevant. TRIAL REGISTRATION: Current Controlled Trials ISRCTN41458548. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 2. See the NIHR Journals Library website for further project information.

Bowel cancer (also known as colorectal cancer) is the fourth commonest cancer in the UK. When the cancer is confined to the bowel and/or the surrounding lymph nodes (early bowel cancer), it is typically treated with an operation to remove the cancer with or without the addition of chemotherapy. Following this treatment, many patients will be cured, but in approximately one in five patients the cancer may come back (recur) either in the bowel or in another organ (e.g. the liver). Consequently, after treatment of early bowel cancer, clinicians often follow up patients in the hope of detecting any recurrent cancer at an early and treatable stage. For the four out of five patients whose cancer will never recur, this follow-up is unnecessary and burdensome on both the NHS and the patients. Better markers are needed to inform which patients do and do not need to undergo this surveillance. Over the last decade, evidence has accumulated to show that the way that a patient's immune system responds to a cancer influences the likelihood of the cancer recurring. It is plausible that those with the most immune cells in their cancer have such a small chance of recurrence that follow-up is not necessary. To validate this in an accurately followed-up population of patients with bowel cancer, we collected cancer tissue specimens from 701 patients in the Follow-up After Colorectal Surgery (FACS) trial and developed methods to count the number of immune cells in their cancers. At present, methods are still under development to automate the process. Indeed, if this were ever to become part of routine practice in NHS laboratories, then automation would be essential.

CTEN Induces Tumour Cell Invasion and Survival and Is Prognostic in Radiotherapy-Treated Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is a heterogenous disease treated with surgery and/or (chemo) radiotherapy, but up to 50% of patients with late-stage disease develop locoregional recurrence. Determining the mechanisms underpinning treatment resistance could identify new therapeutic targets and aid treatment selection. C-terminal tensin-like (CTEN) is a member of the tensin family, upregulated in several cancers, although its expression and function in HNSCC are unknown. We found that CTEN is commonly upregulated in HNSCC, particularly HPV-ve tumours. In vitro CTEN was upregulated in HPV-ve (n = 5) and HPV+ve (n = 2) HNSCC cell lines. Stable shRNA knockdown of CTEN in vivo significantly reduced tumour growth (SCC-25), and functional analyses in vitro showed that CTEN promoted tumour cell invasion, colony formation and growth in 3D-culture (SCC-25, Detroit 562). RNA sequencing of SCC-25 cells following CTEN siRNA knockdown identified 349 differentially expressed genes (logFC > 1, p < 0.05). Gene ontology analysis highlighted terms relating to cell locomotion and apoptosis, consistent with in vitro findings. A membrane-based antibody array confirmed that CTEN regulated multiple apoptosis-associated proteins, including HSP60 and cleaved caspase-3. Notably, in a mixed cohort of HPV+ve and HPV-ve HNSCC patients (n = 259), we found a significant, independent negative association of CTEN with prognosis, limited to those patients treated with (chemo)radiotherapy, not surgery, irrespective of human papillomavirus (HPV) status. These data show that CTEN is commonly upregulated in HNSCC and exerts several functional effects. Its potential role in modulating apoptotic response to therapy suggests utility as a predictive biomarker or radio-sensitising target.

Protein kinase C inhibitors override ZEB1-induced chemoresistance in HCC.

Epithelial-mesenchymal transition (EMT) is a process by which tumour cells lose epithelial characteristics, become mesenchymal and highly motile. EMT pathways also induce stem cell features and resistance to apoptosis. Identifying and targeting this pool of tumour cells is a major challenge. Protein kinase C (PKC) inhibition has been shown to eliminate breast cancer stem cells but has never been assessed in hepatocellular cancer (HCC). We investigated ZEB family of EMT inducer expression as a biomarker for metastatic HCC and evaluated the efficacy of PKC inhibitors for HCC treatment. We showed that ZEB1 positivity predicted patient survival in multiple cohorts and also validated as an independent biomarker of HCC metastasis. ZEB1-expressing HCC cell lines became resistant to conventional chemotherapeutic agents and were enriched in CD44high/CD24low cell population. ZEB1- or TGFß-induced EMT increased PKCα abundance. Probing public databases ascertained a positive association of ZEB1 and PKCα expression in human HCC tumours. Inhibition of PKCα activity by small molecule inhibitors or by PKCA knockdown reduced viability of mesenchymal HCC cells in vitro and in vivo. Our results suggest that ZEB1 expression predicts survival and metastatic potential of HCC. Chemoresistant/mesenchymal HCC cells become addicted to PKC pathway and display sensitivity to PKC inhibitors such as UCN-01. Stratifying patients according to ZEB1 and combining UCN-01 with conventional chemotherapy may be an advantageous chemotherapeutic strategy.

Breast cancer in patients with germline TP53 pathogenic variants have typical tumour characteristics: the Cohort study of TP53 carrier early onset breast cancer (COPE study).

Germline TP53 pathogenic variants are rare but associated with a high risk of cancer; they are often identified in the context of clinically diagnosed Li-Fraumeni syndrome predisposing to a range of young onset cancers including sarcomas and breast cancer. The study aim was to conduct a detailed morphological review and immuno-phenotyping of breast cancer arising in carriers of a germline TP53 pathogenic variant. We compared breast cancers from five defined groups: (1) TP53 carriers with breast cancer (n = 59), (2) early onset HER2-amplified breast cancer, no germline pathogenic variant in BRCA1/2 or TP53 (n = 55), (3) BRCA1 pathogenic variant carriers (n = 60); (4) BRCA2 pathogenic variant carriers (n = 61) and (5) young onset breast cancer with no known germline pathogenic variant (n = 98). Pathologists assessed a pre-agreed set of morphological characteristics using light microscopy. Immunohistochemistry (IHC) for HER2, ER, PR, p53, integrin alpha v beta 6 (αvß6) integrin, α-smooth muscle actin (α-SMA) and pSMAD2/3 was performed on tissue microarrays of invasive carcinoma. We confirmed a previously reported high prevalence of HER2-amplified, ductal no special type invasive breast carcinoma amongst known TP53 germline pathogenic variant carriers 20 of 36 (56%). Furthermore we observed a high frequency of densely sclerotic tumour stroma in cancers from TP53 carriers (29/36, 80.6%) when compared with non-carriers, 50.9% (28/55), 34.7% (50/144), 41.4% (65/157), 43.8% (95/217) in groups 2-5 respectively. The majority of germline TP53 gene carrier breast tumours had a high intensity of integrin αvß6, α-SMA and pSMAD2/3 expression in the majority of cancer cells. In conclusion, aggressive HER2 positive breast cancers with densely sclerotic stroma are common in germline TP53 carriers. High levels of αvß6 integrin, α-SMA and pSMAD2/3 expression suggest that the dense stromal phenotype may be driven by upregulated transforming growth factor beta signalling.

HPV, tumour metabolism and novel target identification in head and neck squamous cell carcinoma.

BACKGROUND: Metabolic changes in tumour cells are used in clinical imaging and may provide potential therapeutic targets. Human papillomavirus (HPV) status is important in classifying head and neck cancers (HNSCC), identifying a distinct clinical phenotype; metabolic differences between these HNSCC subtypes remain poorly understood. METHODS: We used RNA sequencing to classify the metabolic expression profiles of HPV+ve and HPV-ve HNSCC, performed a meta-analysis on FDG-PET imaging characteristics and correlated results with in vitro extracellular flux analysis of HPV-ve and HPV+ve HNSCC cell lines. The monocarboxylic acid transporter-1 (MCT1) was identified as a potential metabolic target and tested in functional assays. RESULTS: Specific metabolic profiles were associated with HPV status, not limited to carbohydrate metabolism. There was dominance of all energy pathways in HPV-negative disease, with elevated expression of genes associated with glycolysis and oxidative phosphorylation. In vitro analysis confirmed comparative increased rates of oxidative phosphorylation and glycolysis in HPV-negative cell lines. PET SUV(max) scores however were unable to reliably differentiate between HPV-positive and HPV-negative tumours. MCT1 expression was significantly increased in HPV-negative tumours, and inhibition suppressed tumour cell invasion, colony formation and promoted radiosensitivity. CONCLUSION: HPV-positive and negative HNSCC have different metabolic profiles which may have potential therapeutic applications.

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays.

AIMS: Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses. METHODS: Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH. RESULTS: Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene. CONCLUSIONS: This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

Assessment of Nuclear ZEB2 as a Biomarker for Colorectal Cancer Outcome and TNM Risk Stratification.

Importance: At present, patients with colorectal cancer (CRC) are risk stratified using TNM histologic features. More recently, an association between a mesenchymal phenotype and a high risk of disease recurrence and micrometastases has been recognized. Objective: To investigate the association of the epithelial to mesenchymal transition (EMT)-inducing transcription factor ZEB2 (zinc finger E box-binding homeobox 2), survival outcomes, and the efficacy of ZEB2 as a biomarker when added as refinement to TNM staging after curative intent surgery for CRC. Design, Setting, and Participants: ZEB2 expression was assessed using a previously validated scoring system as part of a prospective, observational, masked diagnostic study from January 1, 2008, to December 31, 2013. Data were prospectively collected and analyzed for association with oncologic outcomes from January 1, 2017, to December 31, 2018. An initial test cohort from an academic university medical center of 126 consecutive patients with CRC and, subsequently, an independent validation cohort of 210 patients were examined. ZEB2 positivity was scored by 2 independent, masked pathologists. External validity was tested using an open access gene expression portal. Nomograms were developed with or without ZEB2. Main Outcomes and Measures: Systemic and local recurrence of CRC. Results: The test cohort consisted of 126 consecutive patients (mean [SD] age, 72.7 [11.7] years; 61 [48.4%] male) and the validation cohort of 210 patients (mean [SD] age, 72.0 [10.6] years; 111 [52.9%] male). A total of 52 tumors (41.3%) in the test cohort and 104 (49.5%) in the validation cohort were scored nuclear ZEB2 positive. Survival analysis by the log-rank test found that ZEB2 expression was associated with a significant reduction in overall survival and disease-free survival in both cohorts. Cox proportional hazards regression analysis highlighted ZEB2 as an independent biomarker of shorter overall survival and disease-free survival. Analysis of node-negative disease (n = 222) identified ZEB2 as an independent biomarker of early recurrence and reduced survival. External validation confirmed these findings. Addition of ZEB2 expression to nomograms composed of conventional TNM risk factors improved the ability to identify patients at high risk of recurrence demonstrated by the improvement in concordance index in both test (0.73 to 0.77) and validation (0.82 to 0.87) cohorts. Conclusions and Relevance: The findings suggest that expression of ZEB2 is associated with poor oncologic outcome and distant recurrence. The study also found that the addition of ZEB2 to existing TNM classification improved the ability to stratify patients for risk of recurrence. The results of this study suggest that addition of ZEB2 expression status to the TNM staging system improves the ability to stratify patients at high risk of recurrence.

Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4.

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

Association between miR-31-3p expression and cetuximab efficacy in patients with KRAS wild-type metastatic colorectal cancer: a post-hoc analysis of the New EPOC trial.

BACKGROUND: High miR-31-3p expression is associated with inferior outcomes in KRAS wild-type (WT) advanced colorectal cancer patients treated with anti-EGFR therapy. This study evaluated miR-31-3p expression in patients with operable colorectal liver metastases (LM) enrolled in the New EPOC study. METHODS: MiR-31-3p expression was measured in primary tumors (PT) from 149 KRAS WT patients including 71 receiving chemotherapy alone (CT) and 78 receiving chemotherapy plus cetuximab (CTX). Each treatment arm was split into tertiles based on miR-31-3p expression levels. MiR-31-3p expression was also measured in LM from 94 patients with tumor tissue available. RESULTS: The median progression-free survival for the combined populations with mid or high miR-31-3p expression was shorter in the CTX versus the CT arm (26.7 months versus 12.3 months, HR=2.28 95%CI 1.27; 4.09 p=0.006). Low miR-31-3p expressers had similar outcomes irrespective of treatment (HR=1.06 95%CI 0.43; 2.61 p=0.9). MiR-31-3p expression was correlated between paired PT and LM samples in the CT group but not in the CTX group. CONCLUSIONS: Patients with low miR-31-3p expression in the New EPOC study were not harmed by the addition of cetuximab. This supports miR-31-3p as a promising predictive biomarker for anti-EGFR therapy in KRAS WT advanced colorectal cancer.