E-cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase, aurora, and HSP90 in preclinical models.

Cell-cell and cell-matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial-to-mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E-cadherin (ECAD), integrin ß4 (ITGB4), zonula occludens 1 (ZO-1), and cytokeratins in a single-hospital series of Norwegian patients with colorectal cancer (CRC) stages I-IV (n = 922) using multiplex fluorescence-based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO-1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E-cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Increased sensitivity to SMAC mimetic LCL161 identified by longitudinal ex vivo pharmacogenomics of recurrent, KRAS mutated rectal cancer liver metastases.

Tumor heterogeneity is a primary cause of treatment failure. However, changes in drug sensitivity over time are not well mapped in cancer. Patient-derived organoids (PDOs) may predict clinical drug responses ex vivo and offer an opportunity to evaluate novel treatment strategies in a personalized fashion. Here we have evaluated spatio-temporal functional and molecular dynamics of five PDO models established after hepatic re-resections and neoadjuvant combination chemotherapies in a patient with microsatellite stable and KRAS mutated metastatic rectal cancer. Histopathological differentiation phenotypes of the PDOs corresponded with the liver metastases, and ex vivo drug sensitivities generally reflected clinical responses and selection pressure, assessed in comparison to a reference data set of PDOs from metastatic colorectal cancers. PDOs from the initial versus the two recurrent metastatic settings showed heterogeneous cell morphologies, protein marker expression, and drug sensitivities. Exploratory analyses of a drug screen library of 33 investigational anticancer agents showed the strongest ex vivo sensitivity to the SMAC mimetic LCL161 in PDOs of recurrent disease compared to those of the initial metastasis. Functional analyses confirmed target inhibition and apoptosis induction in the LCL161 sensitive PDOs from the recurrent metastases. Gene expression analyses indicated an association between LCL161 sensitivity and tumor necrosis factor alpha signaling and RIPK1 gene expression. In conclusion, LCL161 was identified as a possible experimental therapy of a metastatic rectal cancer that relapsed after hepatic resection and standard systemic treatment.

The expressed mutational landscape of microsatellite stable colorectal cancers.

BACKGROUND: Colorectal cancer is the 2nd leading cause of cancer-related deaths with few patients benefiting from biomarker-guided therapy. Mutation expression is essential for accurate interpretation of mutations as biomarkers, but surprisingly, little has been done to analyze somatic cancer mutations on the expression level. We report a large-scale analysis of allele-specific mutation expression. METHODS: Whole-exome and total RNA sequencing was performed on 137 samples from 121 microsatellite stable colorectal cancers, including multiregional samples of primary and metastatic tumors from 4 patients. Data were integrated with allele-specific resolution. Results were validated in an independent set of 241 colon cancers. Therapeutic associations were explored by pharmacogenomic profiling of 15 cell lines or patient-derived organoids. RESULTS: The median proportion of expressed mutations per tumor was 34%. Cancer-critical mutations had the highest expression frequency (gene-wise mean of 58%), independent of frequent allelic imbalance. Systematic deviation from the general pattern of expression levels according to allelic frequencies was detected, including preferential expression of mutated alleles dependent on the mutation type and target gene. Translational relevance was suggested by correlations of KRAS/NRAS or TP53 mutation expression levels with downstream oncogenic signatures (p < 0.03), overall survival among patients with stage II and III cancer (KRAS/NRAS: hazard ratio 6.1, p = 0.0070), and targeted drug sensitivity. The latter was demonstrated for EGFR and MDM2 inhibition in pre-clinical models. CONCLUSIONS: Only a subset of mutations in microsatellite stable colorectal cancers were expressed, and the "expressed mutation dose" may provide an opportunity for more fine-tuned biomarker interpretations.

De novo transcriptomic subtyping of colorectal cancer liver metastases in the context of tumor heterogeneity.

BACKGROUND: Gene expression-based subtyping has the potential to form a new paradigm for stratified treatment of colorectal cancer. However, current frameworks are based on the transcriptomic profiles of primary tumors, and metastatic heterogeneity is a challenge. Here we aimed to develop a de novo metastasis-oriented framework. METHODS: In total, 829 transcriptomic profiles from patients with colorectal cancer were analyzed, including primary tumors, liver metastases, and non-malignant liver samples. High-resolution microarray gene expression profiling was performed of 283 liver metastases from 171 patients treated by hepatic resection, including multiregional and/or multi-metastatic samples from each of 47 patients. A single randomly selected liver metastasis sample from each patient was used for unsupervised subtype discovery by nonnegative matrix factorization, and a random forest prediction model was trained to classify multi-metastatic samples, as well as liver metastases from two independent series of 308 additional patients. RESULTS: Initial comparisons with non-malignant liver samples and primary colorectal tumors showed a highly variable degree of influence from the liver microenvironment in metastases, which contributed to inter-metastatic transcriptomic heterogeneity, but did not define subtype distinctions. The de novo liver metastasis subtype (LMS) framework recapitulated the main distinction between epithelial-like and mesenchymal-like tumors, with a strong immune and stromal component only in the latter. We also identified biologically distinct epithelial-like subtypes originating from different progenitor cell types. LMS1 metastases had several transcriptomic features of cancer aggressiveness, including secretory progenitor cell origin, oncogenic addictions, and microsatellite instability in a microsatellite stable background, as well as frequent RAS/TP53 co-mutations. The poor-prognostic association of LMS1 metastases was independent of mutation status, clinicopathological variables, and current subtyping frameworks (consensus molecular subtypes and colorectal cancer intrinsic subtypes). LMS1 was also the least heterogeneous subtype in comparisons of multiple metastases per patient, and tumor heterogeneity did not confound the prognostic value of LMS1. CONCLUSIONS: We report the first large study of multi-metastatic gene expression profiling of colorectal cancer. The new metastasis-oriented subtyping framework showed potential for clinically relevant transcriptomic classification in the context of metastatic heterogeneity, and an LMS1 mini-classifier was constructed to facilitate prognostic stratification and further clinical testing.

Metastatic heterogeneity of the consensus molecular subtypes of colorectal cancer.

Gene expression-based subtypes of colorectal cancer have clinical relevance, but the representativeness of primary tumors and the consensus molecular subtypes (CMS) for metastatic cancers is not well known. We investigated the metastatic heterogeneity of CMS. The best approach to subtype translation was delineated by comparisons of transcriptomic profiles from 317 primary tumors and 295 liver metastases, including multi-metastatic samples from 45 patients and 14 primary-metastasis sets. Associations were validated in an external data set (n = 618). Projection of metastases onto principal components of primary tumors showed that metastases were depleted of CMS1-immune/CMS3-metabolic signals, enriched for CMS4-mesenchymal/stromal signals, and heavily influenced by the microenvironment. The tailored CMS classifier (available in an updated version of the R package CMScaller) therefore implemented an approach to regress out the liver tissue background. The majority of classified metastases were either CMS2 or CMS4. Nonetheless, subtype switching and inter-metastatic CMS heterogeneity were frequent and increased with sampling intensity. Poor-prognostic value of CMS1/3 metastases was consistent in the context of intra-patient tumor heterogeneity.

Molecular correlates of sensitivity to PARP inhibition beyond homologous recombination deficiency in pre-clinical models of colorectal cancer point to wild-type TP53 activity.

BACKGROUND: PARP inhibitors are active in various tumour types beyond BRCA-mutant cancers, but their activity and molecular correlates in colorectal cancer (CRC) are not well studied. METHODS: Mutations and genome-wide mutational patterns associated with homologous recombination deficiency (HRD) were investigated in 255 primary CRCs with whole-exome sequencing and/or DNA copy number data. Efficacy of five PARP inhibitors and their molecular correlates were evaluated in 93 CRC cell lines partly annotated with mutational-, DNA copy number-, and/or gene expression profiles. Post-treatment gene expression profiling and specific protein expression analyses were performed in two pairs of PARP inhibitor sensitive and resistant cell lines. FINDINGS: A subset of microsatellite stable (MSS) CRCs had truncating mutations in homologous recombination-related genes, but these were not associated with genomic signatures of HRD. Eight CRC cell lines (9%) were sensitive to PARP inhibition, but sensitivity was not predicted by HRD-related genomic and transcriptomic signatures. In contrast, drug sensitivity in MSS cell lines was strongly associated with TP53 wild-type status (odds ratio 15.7, p = 0.023) and TP53-related expression signatures. Increased downstream TP53 activity was among the primary response mechanisms, and TP53 inhibition antagonized the effect of PARP inhibitors. Wild-type TP53-mediated suppression of RAD51 was identified as a possible mechanism of action for sensitivity to PARP inhibition. INTERPRETATION: PARP inhibitors are active in a subset of CRC cell lines and preserved TP53 function may increase the likelihood of response.

Patient-Derived Organoids from Multiple Colorectal Cancer Liver Metastases Reveal Moderate Intra-patient Pharmacotranscriptomic Heterogeneity.

PURPOSE: Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer. EXPERIMENTAL DESIGN: We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients. RESULTS: Three drug-response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2-5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression. CONCLUSIONS: Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression-based predictive signatures to guide experimental therapies.

Exploratory analyses of consensus molecular subtype-dependent associations of TP53 mutations with immunomodulation and prognosis in colorectal cancer.

BACKGROUND: Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC). MATERIALS AND METHODS: In a single-hospital series of 401 stage I-IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). RESULTS: TP53 mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2-4>0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistry-based quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028). CONCLUSIONS: Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation.

Transcriptional and functional consequences of TP53 splice mutations in colorectal cancer.

TP53 mutations are common in colorectal cancer (CRC). Most TP53 sequencing studies have been restricted to coding regions, but recent studies have revealed that splice mutations can generate transcript variants with distinct tumorigenic and prognostic properties. Here, we performed unrestricted sequencing of all coding sequences and splice regions of TP53 in a single-hospital series of 401 primary CRCs. TP53 splice mutations were detected in 4% of the cases (N = 16), considerably more frequent than reported in major databases, and they were mutually exclusive to exon mutations. RNA sequencing revealed high-level expression of aberrant transcript variants in the majority of splice mutated tumors (75%). Most variants were predicted to produce truncated TP53 proteins, including one sample expressing the potentially oncogenic and druggable p53ψ isoform. Despite heterogeneous transcript structures, downstream transcriptional profiling revealed that TP53 splice mutations had similar effects on TP53 target gene expression and pathway activity as exonic mutations. Intriguingly, TP53 splice mutations were associated with worse 5-year relapse-free survival in stage II disease, compared to both TP53 wild-type and exon mutations (P = 0.007). These data highlight the importance of including splice regions when examining the biological and clinical consequences of TP53 mutations in CRC.

Long noncoding RNA MIR31HG is a bona fide prognostic marker with colorectal cancer cell-intrinsic properties.

Elevated miR-31 expression is associated with poor outcome in colorectal cancer (CRC). Whether the prognostic information is independent of known molecular subgroups and gene expression-based consensus molecular subtypes (CMS) is currently unknown. To investigate this, we analyzed nearly 2000 CRC biopsies and preclinical models. The expression of miR-31-5p and its host transcript, long noncoding RNA MIR31HG, was strongly correlated (Spearman's ρ > 0.80). MIR31HG outlier expression was observed in 158/1265 (12%) of pCRCs and was associated with depletion of CMS2-canonical subgroup (odds ratio = 0.21 [0.11-0.35]) and shorter relapse-free survival (RFS) in multivariable analysis (adjusted hazard ratio = 2.2 [1.6-3.0]). For stage II disease, 5-year RFS for patients with MIR31HG outlier status was 49% compared to 77% for those with normal-like expression. MIR31HG outlier status was associated with inferior outcome also within clinical high risk groups and within the poor prognostic CMS4-mesenchymal gene expression subtype specifically. Preclinical models with MIR31HG outlier expression were characterized by reduced expression of MYC targets as well as elevated epithelial-mesenchymal transition, TNF-α/NFκB, TGF-ß, and IFN-α/γ gene expression signatures, indicating cancer cell-intrinsic properties resembling the CMS4 subgroup-associations which were recapitulated in patient biopsies. Moreover, the prognostic value of MIR31HG outlier status was independent of cytotoxic T lymphocyte and fibroblast infiltration. We here present evidence that MIR31HG expression provides clinical stratification beyond major gene expression phenotypes and tumor immune and stromal cell infiltration and propose a model where increased expression is an indicator of a cellular state conferring intrinsic invasive and/or immuno-evasive capabilities.

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer.

We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I-IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5-year relapse-free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I-III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF-mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5-year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2-negative cell lines were significantly more sensitive to chemotherapeutics than CDX2-positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2-negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early-stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF.

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.

CMScaller: an R package for consensus molecular subtyping of colorectal cancer pre-clinical models.

Colorectal cancers (CRCs) can be divided into four gene expression-based biologically distinct consensus molecular subtypes (CMS). This classification provides a potential framework for stratified treatment, but to identify novel CMS-drug associations, translation of the subtypes to pre-clinical models is essential. The currently available classifier is dependent on gene expression signals from the immune and stromal compartments of tumors and fails to identify the poor-prognostic CMS4-mesenchymal group in immortalized cell lines, patient-derived organoids and xenografts. To address this, we present a novel CMS classifier based on a filtered set of cancer cell-intrinsic, subtype-enriched gene expression markers. This new classifier, referred to as CMScaller, recapitulated the subtypes in both in vitro and in vivo models (551 in total). Importantly, by analyzing public drug response data from patient-derived xenografts and cell lines, we show that the subtypes are predictive of response to standard CRC drugs. CMScaller is available as an R package.

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

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.

Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies.

BACKGROUND: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. METHODS: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. RESULTS: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFß signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately » of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. CONCLUSIONS: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

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.