Peter W. Eide

Epigenetic and genetic features of 24 colon cancer cell lines

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research.

Portrait of the PI3K/AKT pathway in colorectal cancer ☆

PI3K/AKT signaling leads to reduced apoptosis, stimulates cell growth and increases proliferation. Under normal conditions, PI3K/AKT activation is tightly controlled and dependent on both extracellular growth signals and the availability of amino acids and glucose. Genetic aberrations leading to PI3K/AKT hyper-activation are observed at considerable frequency in all major nodes in most tumors. In colorectal cancer the most commonly observed pathway changes are IGF2 overexpression, PIK3CA mutations and PTEN mutations and deletions. Combined, these alterations are found in about 40% of large bowel tumors. In addition, but not mutually exclusive to these, KRAS mutations are observed at a similar frequency. There are however additional, less frequent and more poorly understood events that may also push the PI3K/AKT pathway into overdrive and thus promote malignant growth. Here we discuss aberrations of components at the genetic, epigenetic, transcriptional, post-transcriptional, translational and post-translational level where perturbations may drive excessive PI3K/AKT signaling. Integrating multiple molecular levels will advance our understanding of this cancer critical circuit and more importantly, improve our ability to pharmacologically target the pathway in view of clonal development, tumor heterogeneity and drug resistance mechanisms. In this review, we revisit the PI3K/AKT pathway cancer susceptibility syndromes, summarize the known aberrations at the different regulatory levels and the prognostic and predictive values of these alterations in colorectal cancer.

MicroRNAs as growth regulators, their function and biomarker status in colorectal cancer.

Gene expression is in part regulated by microRNAs (miRNAs). This review summarizes the current knowledge of miRNAs in colorectal cancer (CRC); their role as growth regulators, the mechanisms that regulate the miRNAs themselves and the potential of miRNAs as biomarkers. Although thousands of tissue samples and bodily fluids from CRC patients have been investigated for biomarker potential of miRNAs (>160 papers presented in a comprehensive tables), none single miRNA nor miRNA expression signatures are in clinical use for this disease. More than 500 miRNA-target pairs have been identified in CRC and we discuss how these regulatory nodes interconnect and affect signaling pathways in CRC progression.

NEDD4 is overexpressed in colorectal cancer and promotes colonic cell growth independently of the PI3K/PTEN/AKT pathway

Ubiquitination controls multiple cellular processes relevant to cancer pathogenesis. Using Gene Set Enrichment Analysis of an mRNA transcriptome dataset, we have identified genes encoding components of the ubiquitin system that are differentially expressed in colorectal cancers as compared to normal colonic mucosa. Among the significantly overexpressed genes was NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), the prototype member of the HECT (homologous to E6AP C-terminus) E3 ubiquitin ligase family. Previous studies have shown that NEDD4 may act as an oncoprotein by inducing ubiquitination and degradation of the tumor suppressor protein PTEN (phosphatase and tensin homolog). To investigate its functional importance in colorectal cancer, HCT-15 and LoVo colon cancer cells were depleted of NEDD4 by small interfering RNA. The depletion resulted in reduced growth and altered cell morphology in both cell lines. However, NEDD4 depletion did not affect the PTEN protein level or PI3K/AKT signaling pathway activation. Moreover, ectopic expression of NEDD4 did not influence the PTEN subcellular localization or protein level. Collectively, these data demonstrate that NEDD4 is overexpressed in colorectal cancers, and suggest that NEDD4 promotes growth of colon cancer cells independently of PTEN and PI3K/AKT signaling.

The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions

ABSTRACT Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. Highlighted Article: The proto-oncogenic E3 ubiquitin ligase NEDD4 induces ubiquitylation and lysosomal degradation of connexin 43, a protein that is often dysregulated during cancer development, resulting in loss of gap junctions in human carcinoma cells.

Colorectal Cancer Consensus Molecular Subtypes Translated to Preclinical Models Uncover Potentially Targetable Cancer Cell Dependencies

Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression–based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models. Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell–intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities. Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell–adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model. Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. Clin Cancer Res; 24(4); 794–806. ©2017 AACR.

PO-127 Role of the NEDD4 family of E3 ubiquitin ligases in colorectal cancer pathogenesis and their potential as biomarkers

Introduction Colorectal cancer (CRC) is a global health challenge. There is limited response to standard oncological treatment and few stratified treatment options based on prognostic and/or predictive factors. The four biologically distinct and gene expression-based consensus molecular subtypes (CMS) provide prognostic stratification independent of cancer stage, and represent a new potential paradigm for stratified treatment. In this project, we are analysing the biomarker and drug target potential of members of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases in CRC. Material and methods A total of 412 primary CRCs and 51 normal colonic mucosa samples were subjected to genome wide expression analysis at exon level resolution using either the Affymetrix Human Exon Array or the Affymetrix Human Transcriptome Array. The CRISPR/Cas9 system was applied to generate NEDD4 knock-out Caco2 cell lines. Results and discussions We identified NEDD4 (neural precursor cell-expressed developmentally down-regulated 4) as significantly overexpressed in CRC, while the NEDD4 homolog NEDD4L was significantly down regulated. Furthermore, we found that NEDD4 is differentially expressed between the different CMS classes, with high expression particularly in the epithelial and canonical CMS2 group. CRISPR/Cas9-mediated knock-out of NEDD4 in the CRC cell line Caco2 was found to result in altered growth characteristics. Conclusion The data indicate that NEDD4 and NEDD4L are differently expressed in CRC samples compared to normal colonic mucosa, and that knock-out of NEDD4 in Caco2 cells affects their growth characteristics.

Abstract A44: Randomized adapter pools for microRNA sequencing of colorectal cancer cell lines and primary tumors

Single-nucleotide resolution, high throughput and excellent precision make small RNA sequencing ideally suited for addressing a wide array of microRNA (miRNA) research questions. It is however known that adapter ligation introduces bias distorting the relation between sample expression levels and sequencing read counts. To investigate how this issue may affect studies aiming to identify miRNA biomarkers, we utilized the recently published High Definition library preparation protocol to generate miRNA profiles of 35 colorectal cancer cell lines, 55 primary cancer biopsies and 5 adjacent normal mucosa samples. The protocol minimizes ligation bias by means of randomized adapter pools. Comparing the resulting data against publicly available colorectal cancer miRNA datasets, we demonstrate that the High Definition protocol moderates read count distributions, increases accuracy and improves sensitivity while maintaining high reproducibility. Moreover, by taking advantage of the more quantitative read counts, the cancer cell line panel and the normal mucosa samples, we identify a set of miRNAs that are highly expressed in epithelial cancerous cells, deregulated in colorectal cancer as compared to normal mucosa and differentially expressed between established molecular subgroups. As such, these miRNAs present strong candidates for functional validation and biomarker testing. In conclusion, we here present High Definition miRNA sequencing data for a large panel of clinically relevant colorectal cancer samples highlighting the potential of improved small RNA library preparation methods. Citation Format: Peter W. Eide, Lina Cekaite, Ping Xu, Leonardo A. Meza-Zepeda, Tamas Dalmay, Ragnhild A. Lothe. Randomized adapter pools for microRNA sequencing of colorectal cancer cell lines and primary tumors. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A44.