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Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo .

Colorectal cancer (CRC) is a heterogeneous disease that can be classified into distinct molecular subtypes. The aims of this study were 1) to compare claudin ( ) gene expression in CRC samples and normal colon mucosa, and then in the different CRC molecular subtypes, and 2) to assess their prognostic value. expression in CRC samples was analyzed using gene expression data for a cohort of 143 primary CRC samples, and compared in the same CRC samples classified into different molecular subtypes (C1 to C6 according to the Marisa's classification, and CMS1 to CMS4 of the consensus classification). Comparison of expression in normal and tumor colon samples was also made on a smaller number of samples. Then, the relationship between expression profiles and overall survival (OS) and progression-free survival was examined. Compared with normal mucosa, and were upregulated, whereas , , , and were downregulated in CRC samples. Variations in expression profiles were observed mainly in the CMS2/C1 and CMS4/C4 subtypes. Overall, expression of or alone had a strong prognostic value that increased when they were associated. In the CMS4/C4 subtypes, lower expressions of , , and were associated with longer OS. Conversely, in the CMS2 and C1 subtypes, low expression was associated with shorter OS and progression-free survival, suggesting a dual role for as a tumor suppressor/promoter in CRC. and had a prognostic value in the CMS2 and C4 subtypes, respectively. This analysis of gene expression profiles and prognostic value in CRC samples classified according to their molecular subtype shows that CRC heterogeneity must be taken into account when assessing potential value as prognostic markers or therapeutic targets.

The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies. In order to form a tumour, cancer cells have to overcome the controls that normally prevent cells from dividing too often. These controls include an enzyme called PP2A, which inhibits cell division by regulating the activity of other proteins. In a normal cell, PP2A may be temporarily deactivated from time to time to enable the cell to divide. However, PP2A is permanently deactivated in many cancer cells, which allows these cells to divide many times in quick succession. A protein called Greatwall is involved in deactivating PP2A in healthy cells, but it was not clear whether increases in Greatwall activity can promote the formation of tumours. Here, Vera et al. use a variety of cell biology techniques to address this question. The experiments show that increasing the amount of Greatwall in human and mouse cells can promote some of the transformations needed for these cells to become cancerous. Also, Greatwall increases the growth of tumours in mice. These effects are caused by the over-activation of a protein called AKT, which is already known to promote the formation of many cancers. Vera et al. show that Greatwall regulates AKT activity using a different pathway to how it influences PP2A activity. Further experiments revealed that many human tumours, especially those from patients with colon cancer, produce excessive amounts of Greatwall protein. Together, these findings show that Greatwall can promote the development of cancer. A future challenge is to understand how this works and to find out whether high levels of Greatwall are a common feature of other human cancers.

Background The irinotecan-induced phosphokinome changes in colorectal cancer (CRC) cells were used to guide the selection of targeted agents to be tested in combination with irinotecan. Methods Phosphokinome profiling with peptide arrays of tumour samples from nude mice xenografted with HT29 cells and treated or not with an effective dose of irinotecan was used to identify signalling pathways activated by irinotecan treatment. Then, drugs targeting these pathways were combined in vitro with irinotecan to test potential synergistic effect. The interactions between these drug combinations were assessed by a dose matrix approach. Confirmation of the most potential combination has been confirmed in vivo in xenografted mice. Results Irinotecan induced in vivo the activation of AKT and MEK1 phosphorylation. The dose matrix approach showed that BKM120 (PI3K inhibitor) and MEK162 (MEK inhibitor) are synergistic in vitro and in vivo with a cytostatic and cytotoxic effect, while combination of BKM120 and irinotecan or MEK162 and irinotecan are only additive or even antagonistic. However, the triple combination of SN38, BKM120 and MEK162 showed a better synergistic effect that BKM120 and MEK162, indicating that the cells need to inhibit both AKT and ERK pathways to become more sensitive to irinotecan-based chemotherapies. Conclusion Analysis of chemotherapy-induced phosphokinome changes helps to elucidate the mechanisms of drug resistance and to guide the selection of targets for combination therapies with synergistic activity.

Background Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. Methods Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The “one-two punch” sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. Results We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3β/Wnt/β-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. Conclusion Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a “one-two punch” approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

Gene-expression profiles from over 1,000 colorectal tumors define six subtypes with specific phenotypical features, responses to therapy and clinical progression. Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor–targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI 1 in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments. In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients. Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60-100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16).

IntroductionCervical cancer is a major global health issue. The standard treatment for locally advanced disease involves radiochemotherapy followed by uterovaginal brachytherapy (UBT). UBT requires several days of hospitalisation and strict bed rest. UBT often induces pain, anxiety, stress, distress and a decline in physical capacity during and after treatment. Previous research suggests that non-pharmacological interventions, such as yoga, may help alleviate these issues. However, few studies have specifically evaluated their effectiveness in reducing stress during UBT. Furthermore, patient education has been shown to facilitate autonomous practice and to improve patient empowerment. This study aims to evaluate the impact of the KYOCOL protocol, which integrates both a physiotherapy-yoga intervention and an educational programme, on perceived stress and its correlates in patients undergoing UBT.Methods and analysisKYOCOL is an ongoing randomised, prospective trial carried out in three French comprehensive cancer centres, using a quantitative approach complemented by a qualitative component. Eighty patients are planned to be randomised (1:1) into a control arm (standard care) or an intervention arm. In the intervention arm, patients will be educated and supervised by a trained physiotherapist in a physiotherapy-yoga programme and will then perform daily autonomous sessions during UBT and for up to 15 days post-treatment. The primary objective is to assess the impact of the KYOCOL intervention compared with standard care during UBT, on perceived stress 15 days post-UBT, using the 10-item Perceived Stress Scale. Secondary objectives include evaluating the safety of the intervention, its effects on stress, pain and fatigue during UBT, and patient adherence to the programme. Qualitative analyses based on semistructured interview surveys will be conducted to gather valuable information and analyse in depth patients’ experiences with the intervention and UBT.Ethics and disseminationThis study was approved by the French ethics committee (Comité de Protection des Personnes Ouest V, reference number 2023-A01491-44) on 22 February 2024 and will be carried out in accordance with the good clinical practice guidelines and the Declaration of Helsinki. The results will be shared with patients and healthcare professionals and published in a peer-reviewed journal.Trial registration numberNCT06263283.

Previously, we classified colorectal cancers (CRCs) into five CRCAssigner (CRCA) subtypes with different prognoses and potential treatment responses, later consolidated into four consensus molecular subtypes (CMS). Here we demonstrate the analytical development and validation of a custom NanoString nCounter platform-based biomarker assay (NanoCRCA) to stratify CRCs into subtypes. To reduce costs, we switched from the standard nCounter protocol to a custom modified protocol. The assay included a reduced 38-gene panel that was selected using an in-house machine-learning pipeline. We applied NanoCRCA to 413 samples from 355 CRC patients. From the fresh frozen samples (n = 237), a subset had matched microarray/RNAseq profiles (n = 47) or formalin-fixed paraffin-embedded (FFPE) samples (n = 58). We also analyzed a further 118 FFPE samples. We compared the assay results with the CMS classifier, different platforms (microarrays/RNAseq) and gene-set classifiers (38 and the original 786 genes). The standard and modified protocols showed high correlation (> 0.88) for gene expression. Technical replicates were highly correlated (> 0.96). NanoCRCA classified fresh frozen and FFPE samples into all five CRCA subtypes with consistent classification of selected matched fresh frozen/FFPE samples. We demonstrate high and significant subtype concordance across protocols (100%), gene sets (95%), platforms (87%) and with CMS subtypes (75%) when evaluated across multiple datasets. Overall, our NanoCRCA assay with further validation may facilitate prospective validation of CRC subtypes in clinical trials and beyond.

IntroductionOsteoarticular pain is experienced by approximately 50% of patients with breast cancer under hormone therapy and can increase the risk of therapy discontinuation. Among complementary therapies, yoga has shown efficacy regarding reduction of fatigue, anxiety, pain due to hormone therapy and inflammation. Personalised patient education programmes increase engagement and motivation, and induce effective behavioural changes. The SKYPE programme, an integrated intervention combining physiotherapy, yoga and patient education, showed promising efficacy on hormone therapy-induced pain in a previous pilot study. In this study, we hypothesised that using theory-based patient education favour learning and practising 15 min of at-home yoga every day to decrease hormone therapy-induced pain.Methods and analysisThis multicentre randomised study will assess the efficacy of the SKYPE programme on pain reduction compared with standard care in patients with breast cancer reporting osteoarticular pain due to hormone therapy. Main secondary objectives will describe pain evolution and characteristics, patient adhesion to yoga sessions and home practice, forward flexibility, quality of life, fatigue, anxiety and compliance to hormone therapy. Patients in the intervention group will participate in 1 weekly educational yoga session of 90 min for 6 weeks, supervised by physiotherapists (period 1). They will also perform daily at-home 15 min yoga sessions for 12 weeks, the total duration of the intervention (periods 1 and 2). Pain will be evaluated during physiotherapy check-ups at baseline (T0), at 6 weeks (T1) and at 12 weeks (T2).Ethics and disseminationThis study was approved by the ethics committee (CPP Ile de France 8 on 22 June 2020). The results will be disseminated to patients and healthcare professionals, and published in a peer-reviewed journal.Trial registration numberNCT04457895.