Prognostic role of tumour-infiltrating lymphocytes and macrophages in relation to MSI, CDX2 and BRAF status: a population-based study of metastatic colorectal cancer patients.

BACKGROUND: Tumour-infiltrating CD3, CD8 lymphocytes and CD68 macrophages are associated with favourable prognosis in localised colorectal cancer, but the effect in metastatic colorectal cancer (mCRC) is not established. METHODS: A Scandinavian population-based cohort of non-resectable mCRC patients was studied. Tissue microarrays (n = 460) were stained with CD3, CD8 and CD68 using fluorescence-based multiplex immunohistochemistry. Associations with clinicopathological variables, overall survival (OS) and progression-free survival were estimated. RESULTS: Two-thirds of microsatellite instable (MSI) and one-fourth of microsatellite stable (MSS) tumours displayed the highest quartile density of CD8. For CD3 high vs low cases, median OS was 20 vs 16 months (HR: 0.76, 95% CI: 0.59, 0.76, p = 0.025) with 3-year OS of 27 vs 13%. For CD68 high vs low cases, median OS was 23 vs 15 months (HR: 0.69, 95% CI: 0.54, 0.88, p = 0.003) with 3-year OS of 28 vs 12%. MSI, BRAF mutation and CDX2 loss were negative prognostic markers independent of tumour immune infiltration. CONCLUSIONS: In mCRC, high lymphocyte infiltration was found in proportions of MSI and MSS tumours-potential subgroups of immunotherapy response. Tumour-infiltrating CD3 lymphocytes and CD68 macrophages were associated with median and long-term survival. MSI was a significant negative prognostic marker despite high immunogenicity.

Spatial analysis and CD25-expression identify regulatory T cells as predictors of a poor prognosis in colorectal cancer.

Regulatory T cells (Tregs) are a heterogeneous cell population that can either suppress or stimulate immune responses. Tumor-infiltrating Tregs are associated with an adverse outcome from most cancer types, but have generally been found to be associated with a good prognosis in colorectal cancer (CRC). We investigated the prognostic heterogeneity of Tregs in CRC by co-expression patterns and spatial analyses with diverse T cell markers, using multiplex fluorescence immunohistochemistry and digital image analysis in two consecutive series of primary CRCs (total n = 1720). Treg infiltration in tumors, scored as FOXP3+ or CD4+/CD25+/FOXP3+ (triple-positive) cells, was strongly correlated to the overall amount of CD3+ and CD8+ T cells, and consequently associated with a favorable 5-year relapse-free survival rate among patients with stage I-III CRC who underwent complete tumor resection. However, high relative expression of the activation marker CD25 in triple-positive Tregs was independently associated with an adverse outcome in a multivariable model incorporating clinicopathological and known molecular prognostic markers (hazard ratio = 1.35, p = 0.028). Furthermore, spatial marker analysis based on Voronoi diagrams and permutation testing of cellular neighborhoods revealed a statistically significant proximity between Tregs and CD8+-cells in 18% of patients, and this was independently associated with a poor survival (multivariable hazard ratio = 1.36, p = 0.017). These results show prognostic heterogeneity of different Treg populations in primary CRC, and highlight the importance of multi-marker and spatial analyses for accurate immunophenotyping of tumors in relation to patient outcome.

Cancer stemness, intratumoral heterogeneity, and immune response across cancers.

Regulatory programs that control the function of stem cells are active in cancer and confer properties that promote progression and therapy resistance. However, the impact of a stem cell-like tumor phenotype ("stemness") on the immunological properties of cancer has not been systematically explored. Using gene-expression-based metrics, we evaluated the association of stemness with immune cell infiltration and genomic, transcriptomic, and clinical parameters across 21 solid cancers. We found pervasive negative associations between cancer stemness and anticancer immunity. This occurred despite high stemness cancers exhibiting increased mutation load, cancer-testis antigen expression, and intratumoral heterogeneity. Stemness was also strongly associated with cell-intrinsic suppression of endogenous retroviruses and type I IFN signaling, and increased expression of multiple therapeutically accessible immunosuppressive pathways. Thus, stemness is not only a fundamental process in cancer progression but may provide a mechanistic link between antigenicity, intratumoral heterogeneity, and immune suppression across cancers.

Digital image analysis of multiplex fluorescence IHC in colorectal cancer recognizes the prognostic value of CDX2 and its negative correlation with SOX2.

Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and ß-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.

A panel of intestinal differentiation markers (CDX2, GPA33, and LI-cadherin) identifies gastric cancer patients with favourable prognosis.

BACKGROUND: Gastric cancer is the fifth most common cancer and the third cause of global cancer mortality. CDX2 is an intestinal differentiation marker with prognostic value in gastric cancer and transcriptionally regulates the expression of glycoprotein A33 (GPA33) and liver intestine cadherin (LI-cadherin). METHODS: This study evaluated the clinical significance of the combined expression of CDX2 and its targets GPA33 and LI-cadherin in gastric cancer by fluorescence-based multiplex immunohistochemistry together with digital image analysis and chromogenic immunohistochemistry in 329 gastric cancer samples arranged in tissue microarrays. Additionally, publicly available RNA-seq expression data from 354 gastric cancer samples from the TCGA database were used to validate the immunohistochemistry results. RESULTS: Expression of the three markers (CDX2, GPA33, and LI-cadherin) was strongly correlated, defining an intestinal differentiation panel. Low or negative protein expression of the intestinal differentiation panel identified patients with particularly poor overall survival, irrespective of the methodology used, and was validated in the independent series at the RNA-seq level. CONCLUSIONS: Expression of the intestinal differentiation panel (CDX2, GPA33, and LI-cadherin) defines a set of biomarkers with a strong biological rationale and favourable impact for prognostication of gastric cancer patients.

Correction: Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage.

BACKGROUND: Intratumoural T-cell infiltrate intensity cortes wrelaith clinical outcome in stage II/III colorectal cancer (CRC). We aimed to determine whether this association varies across this heterogeneous group. METHODS: We performed a pooled analysis of 1804 CRCs from the QUASAR2 and VICTOR trials. Intratumoural CD8+ and CD3+ densities were quantified by immunohistochemistry in tissue microarray (TMA) cores, and their association with clinical outcome analysed by Cox regression. We validated our results using publicly available gene expression data in a pooled analysis of 1375 CRCs from seven independent series. RESULTS: In QUASAR2, intratumoural CD8+ was a stronger predictor of CRC recurrence than CD3+ and showed similar discriminative ability to both markers in combination. Pooled multivariable analysis of both trials showed increasing CD8+ density was associated with reduced recurrence risk independent of confounders including DNA mismatch repair deficiency, POLE mutation and chromosomal instability (multivariable hazard ratio [HR] for each two-fold increase = 0.92, 95%CI = 0.87-0.97, P = 3.6 × 10-3). This association was not uniform across risk strata defined by tumour and nodal stage: absent in low-risk (pT3,N0) cases (HR = 1.03, 95%CI = 0.87-1.21, P = 0.75), modest in intermediate-risk (pT4,N0 or pT1-3,N1-2) cases (HR = 0.92, 95%CI = 0.86-1.0, P = 0.046) and strong in high-risk (pT4,N1-2) cases (HR = 0.87, 95%CI = 0.79-0.97, P = 9.4 × 10-3); PINTERACTION = 0.090. Analysis of tumour CD8A expression in the independent validation cohort revealed similar variation in prognostic value across risk strata (PINTERACTION = 0.048). CONCLUSIONS: The prognostic value of intratumoural CD8+ cell infiltration in stage II/III CRC varies across tumour and nodal risk strata.

Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients.

Semiquantitative assessment of immune markers by immunohistochemistry (IHC) has significant limitations for describing the diversity of the immune response in cancer. Therefore, we evaluated a fluorescence-based multiplexed immunohistochemical method in combination with a multispectral imaging system to quantify immune infiltrates in situ in the environment of non-small-cell lung cancer (NSCLC). A tissue microarray including 57 NSCLC cases was stained with antibodies against CD8, CD20, CD4, FOXP3, CD45RO, and pan-cytokeratin, and immune cells were quantified in epithelial and stromal compartments. The results were compared with those of conventional IHC, and related to corresponding RNA-sequencing (RNAseq) expression values. We found a strong correlation between the visual and digital quantification of lymphocytes for CD45RO (correlation coefficient: r = 0.52), FOXP3 (r = 0.87), CD4 (r = 0.79), CD20 (r = 0.81) and CD8 (r = 0.90) cells. The correlation with RNAseq data for digital quantification (0.35-0.65) was comparable to or better than that for visual quantification (0.38-0.58). Combination of the signals of the five immune markers enabled further subpopulations of lymphocytes to be identified and localized. The specific pattern of immune cell infiltration based either on the spatial distribution (distance between regulatory CD8+ T and cancer cells) or the relationships of lymphocyte subclasses with each other (e.g. cytotoxic/regulatory cell ratio) were associated with patient prognosis. In conclusion, the fluorescence multiplexed immunohistochemical method, based on only one tissue section, provided reliable quantification and localization of immune cells in cancer tissue. The application of this technique to clinical biopsies can provide a basic characterization of immune infiltrates to guide clinical decisions in the era of immunotherapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Connexins in colorectal cancer pathogenesis.

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/ß-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.

A novel transcript, VNN1-AB, as a biomarker for colorectal cancer.

Colorectal cancer is a global health challenge with high incidence rate and mortality. The patients' prognosis is strongly associated with disease stage and currently there is a need for improved prognostic and predictive biomarkers. In this study, novel colorectal cancer-specific transcript structures were nominated from whole transcriptome sequencing of seven colorectal cancer cell lines, two primary colorectal carcinomas with corresponding normal colonic mucosa and 16 normal tissues. The nominated transcripts were combined with gene level outlier expression analyses in a cohort of 505 colorectal cancers to identify biomarkers with capacity to stratify colorectal cancer subgroups. The transcriptome sequencing data and outlier expression analysis revealed 11 novel colorectal cancer-specific exon-exon junctions, of which 3 were located in the gene VNN1. The junctions within VNN1 were further characterized using rapid amplification of cDNA ends (RACE) and the prevalence of the subsequently characterized novel transcript, VNN1-AB, was investigated by real-time RT-PCR in 291 samples of miscellaneous origins. VNN1-AB was not present in any of the 43 normal colorectal tissue samples investigated, but in 5 of the 6 polyps, and 102 of the 136 (75%) colorectal cancers. We have identified a novel transcript of the VNN1 gene, with an organ-confined complete specificity for colorectal neoplasia.

Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43.

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

The prognostic effect of tumor-associated macrophages in stage I-III colorectal cancer depends on T cell infiltration.

BACKGROUND: Tumor-associated macrophages (TAMs) are associated with unfavorable patient prognosis in many cancer types. However, TAMs are a heterogeneous cell population and subsets have been shown to activate tumor-infiltrating T cells and confer a good patient prognosis. Data on the prognostic value of TAMs in colorectal cancer are conflicting. We investigated the prognostic effect of TAMs in relation to tumor-infiltrating T cells in colorectal cancers. METHODS: The TAM markers CD68 and CD163 were analyzed by multiplex fluorescence immunohistochemistry and digital image analysis on tissue microarrays of 1720 primary colorectal cancers. TAM density in the tumor stroma was scored in relation to T cell density (stromal CD3+ and epithelial CD8+ cells) and analyzed in Cox proportional hazards models of 5-year relapse-free survival. Multivariable survival models included clinicopathological factors, MSI status and BRAFV600E mutation status. RESULTS: High TAM density was associated with a favorable 5-year relapse-free survival in a multivariable model of patients with stage I-III tumors (p = 0.004, hazard ratio 0.94, 95% confidence interval 0.90-0.98). However, the prognostic effect was dependent on tumoral T-cell density. High TAM density was associated with a good prognosis in patients who also had high T-cell levels in their tumors, while high TAM density was associated with poorer prognosis in patients with low T-cell levels (pinteraction = 0.0006). This prognostic heterogeneity was found for microsatellite stable tumors separately. CONCLUSIONS: This study supported a phenotypic heterogeneity of TAMs in colorectal cancer, and showed that combined tumor immunophenotyping of multiple immune cell types improved the prediction of patient prognosis.

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.

Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome.

This article is the first to show that loss of connexin43 (Cx43) expression in colorectal tumors is correlated with significantly shorter relapse-free and overall survival. Cx43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis. In addition, Cx43 was found to colocalize with ß-catenin and reduce Wnt signaling. The study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. The study has important implications for the assessment of Cx43 as a prognostic marker and target in colorectal cancer prevention and therapy. Gap junctions consist of intercellular channels that permit direct transfer of ions and small molecules between adjacent cells. The gap junction channel protein Cx43 plays important roles in cell growth control and differentiation and is frequently dysregulated in human cancers. However, the functional importance and clinical relevance of Cx43 in cancer development has remained elusive. Here, we show that Cx43 is downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. The in situ protein expression of Cx43 was analyzed in colorectal tumors in a cohort of 674 patients and related to established clinicopathological variables and survival. A subgroup of the patients had weak or no expression of Cx43 in tumors. Loss of Cx43 expression was significantly correlated with shorter relapse-free and overall survival. Loss of Cx43 further identified a high-risk subgroup among stage I and stage II patients with reduced relapse-free and overall survival. Ectopic expression of Cx43 in the colon cancer cell line HT29 was associated with reduced growth in monolayer and soft agar cultures and in tumor xenografts. Cx43 was found to colocalize with ß-catenin and negatively regulate the Wnt signaling pathway, and expression of Cx43 was associated with increased levels of apoptosis. Altogether, these data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.

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.

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.

Inhibition of connexin 43 gap junction channels by the endocrine disruptor ioxynil.

Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin 43. Connexin 43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin 43 gap junction channels. Both compounds induced rapid loss of connexin 43 gap junctions at the plasma membrane and increased connexin 43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin 43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

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.