Abstract Patients with colorectal cancer have few molecularly guided treatment options. Cancers with exceptional treatment response are valuable models for discovery of molecular response mechanisms and might reveal new predictive biomarkers to guide treatment selection. However, the rarity of events is a clinical challenge. We performed integrated high-throughput drug sensitivity testing ( n = 620 drugs) and molecular analyses of 103 colorectal cancer cell lines to build an in vitro foundation for exceptional treatment responses and their molecular markers. Exceptional responses were scored by a two-way outlier detection approach and were identified in 67 unique cell line-drug pairs (0.13% of all pairs tested). This involved 35 cell lines (34.0%) and 49 drugs (7.9%) representing standard, experimental, and non-oncology drugs of diverse classes. Plausible response mechanisms on the gene, protein, and/or pathway expression levels were identified at baseline in 89.6% of the 67 pairs. Experimental validation confirmed treatment-induced marker suppression in seven of eight selected pairs, including target engagement of kinase inhibitors and downregulation of markers of extended response mechanisms. Recurrent exceptional sensitivity to the same drug in different cell lines involved functionally convergent molecular mechanisms, but rarely precisely the same marker. Most exceptional sensitivities (77.8%) could be predicted with multivariable transcriptomic models. In conclusion, this study provides in vitro support for biomarker-guided prescreening to potentially obtain exceptional treatment response to diverse drugs and therapeutic targets in colorectal cancer. Most exceptional responses have a clear molecular underpinning. The study also provides a large and openly available pharmaco-transcriptomics resource for colorectal cancer cell lines.

Intercellular communication via gap junctions is often lost during cancer development, which may contribute to increased tumor growth and affect how cancer cells respond to radio- and chemotherapy. Gap junction channels comprise transmembrane proteins belonging to the connexin family, of which connexin43 (Cx43) is the most ubiquitously expressed in humans. SMAD ubiquitination regulatory factor 2 (SMURF2), a member of the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) family of E3 ubiquitin ligases, often accumulates in the cytoplasm in cancer cells, where it may display oncogenic properties. Here, we demonstrate that SMURF2 interacts with Cx43 and promotes its ubiquitination in HeLa cells, which is associated with loss of Cx43-based gap junctions and reduced levels of Cx43. Moreover, SMURF2 was found to cooperate with two other NEDD4 family members, NEDD4 and ITCH, to regulate Cx43 ubiquitination and degradation. Simultaneous depletion of these three E3 ubiquitin ligases significantly reduced the Cx43 ubiquitination and degradation rate compared with their single depletion. Their combined knockdown was also found to reduce the ubiquitination and degradation of Cx43 following exposure to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Collectively, these data identify SMURF2 as a negative regulator of gap junctional intercellular communication in cancer cells by inducing the loss of Cx43-based gap junctions. The study also establishes that Cx43 ubiquitination and degradation are controlled by the concurrent participation of multiple E3 ubiquitin ligases, both under basal conditions and in response to TPA exposure.

The BRAFV600E mutation, present in 8 - 10% of colorectal cancers (CRCs), is associated with poor patient outcome and has limited treatment options. Recent studies have shown that mutations in the E3 ubiquitin ligase RNF43 predict response to anti-EGFR/BRAF combinatory therapy in patients with BRAFV600E metastatic CRC. Gene expression analysis of CRCs with the BRAF/RNF43 co-mutation exhibit significantly higher expression levels of MAP2K1 and MAPK3 compared to their wild-type counterparts. This implies an unknown but clinically significant molecular relationship between RNF43 loss-of-function and the MAPK signaling pathway. RNF43 attenuates WNT signaling by promoting the endocytosis and lysosomal degradation of Frizzled receptors to desensitize the cells to WNT ligands. RNF43 itself is in turn negatively regulated by the R-spondin (RSPO) family of proteins. Activating fusions involving RSPOs and inactivating mutations in RNF43 are found in ∼2% and ∼5% of CRCs, respectively. The aim of the present project is to better understand how gene alterations affecting the R-spondin/RNF43 signaling module influence the response to EGFR/BRAF inhibitor combination treatment. We use patient-derived organoids with different genomic alterations in BRAF and RNF43, APC, or RSPO3 as preclinical models to elucidate signaling dynamics before and after treatment with EGFR and BRAF inhibitors alone or in combination. This analysis includes various omics approaches such as mass spectrometry, RNA sequencing and kinome profiling. Here, we present preliminary results from this study, showing how these treatments differentially affect MAPK and WNT signaling depending on the mutational profile of the organoids Jakob M. Stenersen, Max Z. Totland, Luís Nunes, Ina A. Eilertsen, Vilde C. Elster, Tuula A. Nyman, Ragnihild A. Lothe, Anita Sveen, Kushtrim Kryeziu, Edward Leithe. Implications of genomic alterations in RNF43 and RSPO3 for the response of BRAFV600E metastatic colorectal cancer to anti-EGFR+BRAF combinatory therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4604.

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

Supplemental Materials, Figures S1-6, Tables S1-4. Fig. S1. Flow diagram for inclusion of patients in the study. Fig. S2. RNA secondary structure analysis of the 5'UTR RCC2 mutation compared to wild type. Fig. S3. Vector construction overview. Fig. S4. Morphological changes in actin fiber patterns following depletion of RCC2 protein in HCT15 cells. Fig. S5. Subgroup analyses of in situ protein expression of RCC2 for a consecutive CRC series. Fig. S6. Representative electropherograms from fragment analysis of the 5'UTR RCC2 mutation. Table S1. Statistics for relevant parameters for the test series (n = 37). Table S2. Statistics for relevant parameters for the validation series (n = 122). Table S3. Statistics for relevant parameters for the validation series (n = 60, R0 patients). Table S4. Primer and PCR details.

Gap junctions are specialized regions of the plasma membrane containing clusters of channels that provide for the diffusion of ions and small molecules between adjacent cells. A fundamental role of gap junctions is to coordinate the functions of cells in tissues. Cancer pathogenesis is usually associated with loss of intercellular communication mediated by gap junctions, which may affect tumor growth and the response to radio- and chemotherapy. Gap junction channels consist of integral membrane proteins termed connexins. In addition to their canonical roles in cell-cell communication, connexins modulate a range of signal transduction pathways via interactions with proteins such as β-catenin, c-Src, and PTEN. Consequently, connexins can regulate cellular processes such as cell growth, migration, and differentiation through both channel-dependent and independent mechanisms. Gap junctions are dynamic plasma membrane entities, and by modulating the rate at which connexins undergo endocytosis and sorting to lysosomes for degradation, cells can rapidly adjust the level of gap junctions in response to alterations in the intracellular or extracellular milieu. Current experimental evidence indicates that aberrant trafficking of connexins in the endocytic system is intrinsically involved in mediating the loss of gap junctions during carcinogenesis. This review highlights the role played by the endocytic system in controlling connexin degradation, and consequently gap junction levels, and discusses how dysregulation of these processes contributes to the loss of gap junctions during cancer development. We also discuss the therapeutic implications of aberrant endocytic trafficking of connexins in cancer cells.

Abstract Introduction: NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), a member of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases, has been shown to be an important regulator of multiple proteins involved in cancer development, including the tumor suppressor PTEN and the proto-oncogene MDM2. Furthermore, NEDD4 has been shown to be overexpressed and act as an oncogene in multiple cancer types. We have previously shown that NEDD4 is overexpressed in colorectal cancer (CRC) and that it can promote growth of colon cancer cells independently of PTEN and PI3K/AKT signaling. In the present study, we investigated the role of NEDD4 in regulating the PTEN/PI3K pathway and the MDM2/p53 axis in CRC. Materials and Methods: The CRISPR/Cas9 system was applied to generate a Caco2 NEDD4 knockout cell line, and NEDD4 knockdown was performed in LS174T and SW480 cells. Gene expression profiles of 412 primary CRCs, 51 normal mucosa samples, 38 CRC cell lines, as well as the Caco2 parental and NEDD4 knockout cell lines, were generated using exon-resolution Affymetrix Human Exon Arrays or Human Transcriptome Arrays. The NEDD4 expression level was correlated with the mutation status of KRAS, PTEN, TP53, PIK3CA, BRAF and NRAS. Western blotting was used to detect and quantify NEDD4, PTEN and MDM2 protein levels. Results and Discussions: Gene expression analysis of the patient material confirmed our previous study that NEDD4 is significantly upregulated in CRC as compared to normal colonic mucosa. There was no correlation between NEDD4 expression and mutations in KRAS, BRAF, NRAS, PTEN, PIK3CA and TP53. CRISPR/Cas9-mediated NEDD4 knockout in Caco2 cells resulted in a reduction (P <0.01) in both the PTEN and MDM2 protein levels as compared to control cells. Gene set analysis showed that PI3K/AKT/MTOR signaling was upregulated in the NEDD4 knockout cells as compared to control cells. siRNA-mediated depletion of NEDD4 in LS174T cells was associated with reduced levels of MDM2, but did not affect the PTEN protein level. In SW480 cells, depletion of NEDD4 affected neither the MDM2 nor the PTEN protein level. By analyzing the expression of NEDD4, PTEN and MDM2 in 38 CRC cell lines by Western blotting, a positive correlation (P<0.01) was observed between NEDD4 and PTEN protein, while there was no significant correlation between NEDD4 and MDM2. Conclusion: The data show that NEDD4 is significantly upregulated in CRC, and that NEDD4 expression correlates with PTEN expression. The data further suggest that NEDD4 has the ability to regulate the PTEN and MDM2 protein levels in colon cancer cells in a cell line-specific manner. Citation Format: Lars M. Knudsen, Anita Sveen, Christer A. Andreassen, Christian H. Bergsland, Ina A. Eilertsen, Nikoline L. Rasmussen, Max Z. Totland, Peter W. Eide, Jarle Bruun, Ragnhild A. Lothe, Edward Leithe. Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1433.

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes.

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.

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Introduction The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells to directly exchange ions and small molecules. The connexin channels assemble into distinct plasma membrane domains known as gap junctions. Intercellular communication via gap junctions has an important role in regulating cell growth, differentiation and in maintaining tissue homeostasis. The most ubiquitously expressed connexin isoform in human tissues, connexin 43 (Cx43), acts as a tumour suppressor in multiple tissue types and is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the regulation of Cx43 degradation remains poorly understood. Material and methods The cervical cancer cell lines HeLa and C33a were used as model systems. Silencing and ectopic overexpression of proteins were performed by siRNA and plasmid transfection, respectively. The Cx43 ubiquitination status was determined using immunoprecipitation and western blotting. Gap junction intercellular communication (GJIC) was measured using the scrape loading dye transfer assay. Cells were imaged using confocal microscopy. Results and discussions Here, we identify a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family of E3 ubiquitin ligases, termed ITCH, as a novel regulator of Cx43 degradation and GJIC. Depletion of ITCH resulted in increased Cx43 protein levels, gap junction size and GJIC. Ectopic overexpression of ITCH, but not a catalytically inactive ITCH mutant, led to decreased Cx43 protein levels and loss of gap junctions. The data further indicate that ITCH acts in concert with two other members of the NEDD4 family that previously have been shown to regulate Cx43 degradation, termed NEDD4 and SMURF2. Simultaneous depletion of NEDD4, SMURF2, and ITCH by siRNA was found to result in a significantly lower Cx43 ubiquitination level and reduced Cx43 degradation under basal conditions. The triple knock-down of these three E3 ubiquitin ligases was also found to strongly counteract the TPA (12-O-tetradecanoylphorbol 13-acetate)-induced degradation of Cx43. Conclusion These data identify ITCH as a novel regulator of Cx43 degradation and GJIC. The data also indicate that ITCH acts together with NEDD4 and SMURF2 to mediate the basal and TPA-induced turnover of Cx43.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations—the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers.

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.

Communication between adjacent cells can occur via gap junctions (GJ) composed of connexin (Cx) hexamers that allow passage of small molecules. One of the most widely and highly expressed Cxs in human tissues is Cx43, shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane associated protein that can serve as an A-kinase anchoring protein (AKAP) and hold an anchored pool of PKA. Here, we used the liver epithelial cell line IAR20, which expresses Cx43 as the predominant GJ protein, to test the hypothesis that Ezrin may associate with Cx43 in cell types that form stable GJs and serve as an AKAP. Our biochemical and proteomics data indicate that Ezrin associates with Cx43 in epithelial cells. Analyses by confocal immunofluorescence microscopy and proximity ligation assays demonstrate that Ezrin and Cx43 co-localize, together with zonula occludens-1 (ZO-1) and PKA RIα and RIIα, at the cell membrane. Quantitative gap-FRAP experiments show increased GJ intercellular communication after cAMP stimulation. Moreover, loading of cells with the Ht31 peptide that displaces both PKA RIα and RIIα from the AKAP or a peptide that disrupts the Cx43-Ezrin interaction reverts the effect and reduces the level of communication, supporting the hypothesis that in IAR20 cells Ezrin associates with Cx43 (in complex with ZO-1) which places PKA in proximity to Cx43, enabling its phosphorylation and GJ opening.

In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells to directly exchange ions and small molecules. The connexin channels assemble into distinct plasma membrane domains known as gap junctions. Intercellular communication via gap junctions has an important role in regulating cell growth and differentiation, as well as in maintaining tissue homeostasis. Connexin43 (Cx43), the most ubiquitously expressed connexin isoform in human tissues, has been shown to act as a tumor suppressor and is frequently downregulated during cancer development. Cx43 has a short half-life, and modulation of the Cx43 turnover rate represents an important mechanism by which the level of gap junctional intercellular communication is regulated under basal conditions. Moreover, many growth factors, oncogenes, and tumor promoters are potent inducers of Cx43 endocytosis and endolysosomal degradation, resulting in loss of gap junctions. Emerging evidence indicates that the ubiquitin system has a major role in these processes. Recent studies have shown that ubiquitination is also involved in the autophagy-mediated degradation of Cx43 in a process mediated by the proto-oncogenic E3 ubiquitin ligase NEDD4. Moreover, ubiquitination of connexins has been implicated in modulating the level of intercellular communication via gap junctions in response to oxidative stress. This review article provides an overview of our current understanding of the role of the ubiquitin system in the regulation of connexins and discusses how the malfunction of these processes may contribute to the loss of intercellular communication via gap junctions during carcinogenesis.

Abstract Purpose: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. Experimental Design: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). Results: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5′ UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. Conclusions: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays. Clin Cancer Res; 21(16); 3759–70. ©2015 AACR.

The connexins constitute a family of integral membrane proteins that form channels between adjacent cells. These channels are assembled in plasma membrane domains known as gap junctions and enable cells to directly exchange ions and small molecules. Intercellular communication via gap junctions plays important roles in regulating cell growth and differentiation and in maintaining tissue homeostasis. This type of cell communication is often impaired during cancer development, and several members of the connexin protein family have been shown to act as tumor suppressors. Emerging evidence suggests that the connexin protein family has important roles in colorectal cancer development. In the normal colonic epithelial tissue, three connexin isoforms, connexin 26 (Cx26), Cx32 and Cx43, have been shown to be expressed at the protein level. Colorectal cancer development is associated with loss of connexin expression or relocalization of connexins from the plasma membrane to intracellular compartments. Downregulation of connexins in colorectal carcinomas at the transcriptional level involves cancer‐specific promoter hypermethylation. Recent studies suggest that Cx43 may constrain growth of colon cancer cells by interfering with the Wnt/β‐catenin pathway. There is also increasing evidence that the connexins may have potential as prognostic markers in colorectal cancer. This review discusses the role of connexins in colorectal cancer pathogenesis, as well as their potential as prognostic markers and targets in the prevention and treatment of the disease.