Conor Bradley

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

EphA2 expression is a key driver of migration and invasion and a poor prognostic marker in colorectal cancer.

Purpose: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. Experimental Design: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. Results: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. Conclusions: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target. Clin Cancer Res; 22(1); 230–42. ©2015 AACR.

Targeting c-MET in gastrointestinal tumours: rationale, opportunities and challenges

Data from many preclinical studies, including those using cellular models of colorectal, gastric, gastro-oesophageal and gastro-oesophageal junction cancers, indicate that the hepatocyte growth factor (HGF)–hepatocyte growth factor receptor (c-MET) pathway is vital for the growth, survival and invasive potential of gastrointestinal cancers. Following the availability of data from these various studies, and data on c-MET expression as a biomarker that indicates a poor prognosis in patients with gastrointestinal cancer and increased c-MET expression, inhibitors targeting this pathway have entered the clinic in the past decade. However, the design of clinical trials that incorporate the use of HGF/c-MET inhibitors in their most appropriate genetic and molecular context remains crucial. Recognizing and responding to this challenge, the European Commission funded Framework 7 MErCuRIC programme is running a biomarker-enriched clinical trial investigating the efficacy of combined c-MET/MEK inhibition in patients with RAS-mutant or RAS-wild-type metastatic colorectal cancer with aberrant c-MET expression. The design of this trial enables the continued refinement of the predictive biomarker and co-development of companion diagnostics. In this Review, we focus on advances in our understanding of inhibition of the HGF/c-MET pathway in patients with gastro-intestinal cancers, the prominent challenges facing the clinical translation and implementation of agents targeting HGF/c-MET, and discuss the various efforts, and associated obstacles to the discovery and validation of biomarkers that will enable patient stratification in this context.

A calcium-dependent interaction between calmodulin and the calponin homology domain of human IQGAP1

IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium–calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered.

Transcriptional upregulation of c-MET is associated with invasion and tumor budding in colorectal cancer

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

FLIP: a targetable mediator of resistance to radiation in non-small cell lung cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non–small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo. Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432–41. ©2016 AACR.

Corrigendum: Targeting c-MET in gastrointestinal tumours: rationale, opportunities and challenges

This corrects the article DOI: 10.1038/nrclinonc.2017.40

Tracing the steps of cancer evolution

Nature reviews | CliniCal OnCOlOgy Delineating the routes of tumour evolution that define clear cell renal cell carcinoma (ccRCC) could help to identify new treatment approaches and predict patient outcomes. Three papers recently published in Cell now reveal the interim findings of the TRACERx Renal study, which offer insights into the spatial and temporal evolutionary trajectories of RCC. Following previous observations of extensive intratumour heterogeneity (ITH) within RCC, TRACERx Renal is aimed at elucidating the trajectories of tumour evolution that define RCC and explaining how they influence patient outcomes. “To capture the diversity within tumours, we performed very comprehensive sampling of the primary (or metastatic) tumour, taking up to 100 spatially separate biopsies from the surgical specimen,” explains senior investigator Samra Turajlic. Following longitudinal sampling, genomic profiling was performed on each sample to determine the unique evolutionary pathway of each tumour. In the first study, Turajlic et al. analysed 1,206 primary tumour regions from 101 patients and used conserved patterns of mutational ordering, timing and co-occurrence to classify tumours into seven distinct evolutionary subtypes, or three broad categories, which correlate with known clinical phenotypes. “The three categories are distinguished by two features — the number of chromosome alterations and the amount of genetic diversity,” explains Turajlic. The first subtype — comprising aggressive tumours that rapidly grow and metastasize — acquires multiple mutational drivers and chromosomal alterations early in development, but has low ITH. Tumours that gradually accumulate chromosomal damage, which have high ITH, form the second subtype; they grow and metastasize slowly, often forming solitary or oligometastases. Tumours of the third subtype, defined by slow evolution with low ITH, acquisition of few genetic drivers, and low chromosomal instability, have limited metastatic potential. These insights illustrate the potential of such evolutionary classifications as biomarkers for guiding intervention and surveillance. The second study investigated the features selected for in metastatic clones and the routes and timing of metastases across multiple anatomical sites. The analysis included a total of 575 primary and 335 matched metastatic biopsy samples from 100 patients enrolled in TRACERx Renal and 2 validation cohorts. “The clonal populations that are metastasis-competent have high levels of chromosomal damage and, in particular, are enriched for loss of chromosome 9p and 14q,” explains Turajlic. Interestingly, metastases contained fewer subclonal driver alterations than their matched primary tumours, suggesting that metastatic tumours are more homogeneous. In addition, two distinct modes of metastatic dissemination were uncovered. Primary tumours with low ITH and high genomic instability acquired metastatic competence from the most recent common ancestor, leading to rapid progression, whereas those with low genomic instability and high ITH gradually acquired metastatic capacity, leading to attenuated progression. Importantly, the data suggest that metastasis-enabling events (such as 9p and/or 14q loss) could be potential predictive biomarkers. The third study traced the early events leading to RCC in 95 biopsy samples from 33 patients. The most interesting finding was that the initiating driver abnormality, chromosome 3p loss with 5q gain, occurs in childhood and adolescence, 30–50 years before diagnosis. Clonal expansion following chromosome 3p loss results in only a few hundred cells, which, upon acquisition of somatic driver mutations, might lead to sporadic tumours. The authors postulate that the well-defined sequence and timing of these initiating events could lead to new opportunities for early intervention. Overall, these interim findings shed light on the deterministic evolutionary pathways in RCC and could lead to new options for patient management. The next milestone for TRACERx Renal will be the completion of accrual (>300 patients). “Meanwhile, we are looking at other aspects of clinical behaviour such as response to drug therapy in relation to these evolutionary subtypes,” concludes Turajlic. “We are also planning the next steps to validate this approach as a clinical tool for routine use to inform prognosis and help clinical decision-making.” Conor A. Bradley K I D N E Y C A N C E R

Abstract A34: Regulation of EGFR signaling by DRD1 in lung cancer

Dopamine (DA) signaling contributes to a variety of physiologic processes in the central nervous system (CNS), including voluntary movement, reward, sleep, attention, memory and learning. However, emerging work highlights the importance of the DA pathway in non CNS-related functions, including regulation of the cardiovascular, olfactory, endocrine, and immune systems. Recently, our group discovered and replicated an epidemiologic association between an SNP in the 3’ UTR of the DRD1 (dopamine receptor D1) gene and risk of developing lung cancer. Although dopamine mediates nicotine addiction, the association was also observed in never-smokers, suggesting that DRD1 may be directly involved in lung cancer pathogenesis. As there are sparse data regarding the DAP in normal lung, we initially characterized dopamine signaling (receptors, transporters, metabolizing enzymes, synthesis) in the proximal and distal regions of the normal lung. Our results show that the DAP, including DRD1, is expressed throughout the pulmonary system. In lung cancer, DRD1 expression is lost in a majority of cases—most likely due to aberrant methylation—such that patients with high expression have significantly better survival. We generated stable lung cancer cell lines harboring shRNA-mediated knockdown of DRD1 and stable DRD1 overexpression. Downregulation of DRD1 significantly increased cell proliferation as well as xenograft tumor formation, while overexpression of DRD1 led to a suppression of cell growth. Using transcriptome, kinome and phosphoproteome arrays, we also discovered that modulation of DRD1 expression specifically modulates EGFR phosphorylation and MAPK/ERK signaling. Moreover, confocal analyses show that DRD1 co-localizes with EGFR at the cell membrane, suggesting that this could be a direct interaction. Although DRD1 has been linked with regulation of inflammation, we have not find significant similar evidence in this study to date. Our work highlights a novel aspect of DRD1 singaling in the context of lung cancer and possibly normal pulmonary physiology. This study could lead to the identification of novel oncotargets, biomarkers and therapeutic strategies for tumors involving EGFR signaling or mutations in lung cancer. These data also contribute to a preclinical rationale for repositioning FDA-approved antidepressants as adjunct therapeutics with chemotherapy and may have significant translational impact for some lung cancer patients. Indeed, our recent findings show that antidepressants—specifically NDRIs (norepinephrine and dopamine reuptake inhibitors)—have better outcomes. Citation Format: Leila Toulabi, Conor Bradley, Adriana Zingone, Christopher Grant, Oscar Vidal, Khadijah Mitchell, Brid M. Ryan. Regulation of EGFR signaling by DRD1 in lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A34.

Abstract 924: Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal cancer

Background: The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (1-3%), and mutated (1-3%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-dependent and independent activation of c-MET has been associated with increased survival and resistance to targeted therapies. This study aimed to investigate the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Methods: In order to model CRC tumour cell invasion, we have generated invasive CRC subpopulations using Boyden Invasion chambers. To model the CRC microenvironment, we have used a range of co-culture techniques with CRC cells and colon fibroblasts. Migration/invasion was determined using xCELLigence System (Roche). c-MET expression in parental and invasive cell lines was measured using Western blotting and qRT-PCR. c-MET expression in CRC FFPE tissues was measured using IHC and RNAScope®. Results: We identified marked upregulated expression of c-MET at both the protein and transcript levels in our invasive CRC cell line models. Importantly, both parental and invasive subpopulations were found to be inherently dependent on c-MET for migration, as RNAi against c-MET abrogated migration/invasion in both parental and invasive models. We also demonstrated that stimulation of CRC cells with rh-HGF resulted in increased CRC cell migration/invasion. In addition, co-culture of CRC cells with colonic myofibroblasts, resulted in marked increases in migratory and invasive capacity, and this was dependent on HGF/c-MET signaling. Interestingly, stimulation with myofibroblast conditioned medium or HGF promotes rapid degradation of c-MET at the protein level, followed by recycling, while MET transcript remains unaltered, illustrating a dynamic expression of c-MET protein in response to activation. We further showed that MET is transcriptionally upregulated in tumour budding foci at the invasive front of a cohort of stage III CRC tumors. Intriguingly, c-MET protein levels do not correlate with the transcript, most likely due to a similar protein degradation process observed in our aforementioned in vitro models. Conclusions: We show for the first time a key role for transcriptional upregulation of MET as a molecular driver of tumour invasion, both in vitro and in stage III CRC tumours. Citation Format: Conor Bradley, Philip Dunne, Stephen McQuaid, Victoria Bingham, Mark Lawler, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 924.