Therapeutic Strategies for Mesenchymal Type Colorectal Cancer

Colorectal cancer is a heterogeneous disease. We are unable to predict which colorectal cancer patients will develop metastases, and which adjuvant therapy is required to prevent outgrowth of metastases in the individual patient. Analysis of molecular characteristics of colorectal cancer could contribute to improved patient selection and targeted therapies. Based on gene expression profiles, four Consensus Molecular Subtypes (CMS1-4) of CRC are distinguished, which vary in activation of cellular signaling pathways and in the composition of the tumor microenvironment consisting of non-neoplastic cells and extracellular matrix. Besides, the CMSs differ in terms of prognosis and show variable response to adjuvant chemotherapy. The mesenchymal type (CMS4) has the worst prognosis and responds poorly to currently used chemotherapies. Identification of this subtype and development of novel therapies against CMS4 are therefore of utmost importance. The aims of studies described in this thesis were to implement CMS classification in clinical practice, to test novel CMS4-targeted therapies in preclinical models and in a clinical trial, and to study molecular classification and surgical treatment of metastatic colorectal cancer. We made use of organoids (three-dimensional cell culture models of tumors), tissue material from colorectal cancer, and clinical data from patients with colorectal cancer in various stages of the disease. A diagnostic test was developed that allows selection of CMS4 tumors for therapeutic purposes. With this test, multiple regions within a single cancer were tested, and it was found that one tumor can contain multiple molecular subtypes. Furthermore, primary cancers and metastases can differ in molecular subtype, and chemotherapeutic treatment can influence the subtype. Finally, with the use of organoids, two promising novel treatments against poor-prognosis colorectal cancer were discovered. The tyrosine kinase inhibitor dasatinib attenuates invasion of tumor cells driven by the microenvironment and could thus improve treatment of CMS4 cancers. Inhibition of the protein ATR increases the efficacy of intraperitoneal chemotherapy treatment by preventing DNA damage repair in tumor cells. Molecular classification of colorectal cancer can be implemented in clinical practise, and can contribute to the development of novel (subtype-targeted) therapeutic strategies against poor-prognosis colorectal cancer

Therapeutic Strategies for Mesenchymal Type Colorectal Cancer

Colorectal cancer is a heterogeneous disease. We are unable to predict which colorectal cancer patients will develop metastases, and which adjuvant therapy is required to prevent outgrowth of metastases in the individual patient. Analysis of molecular characteristics of colorectal cancer could contribute to improved patient selection and targeted therapies. Based on gene expression profiles, four Consensus Molecular Subtypes (CMS1-4) of CRC are distinguished, which vary in activation of cellular signaling pathways and in the composition of the tumor microenvironment consisting of non-neoplastic cells and extracellular matrix. Besides, the CMSs differ in terms of prognosis and show variable response to adjuvant chemotherapy. The mesenchymal type (CMS4) has the worst prognosis and responds poorly to currently used chemotherapies. Identification of this subtype and development of novel therapies against CMS4 are therefore of utmost importance. The aims of studies described in this thesis were to implement CMS classification in clinical practice, to test novel CMS4-targeted therapies in preclinical models and in a clinical trial, and to study molecular classification and surgical treatment of metastatic colorectal cancer. We made use of organoids (three-dimensional cell culture models of tumors), tissue material from colorectal cancer, and clinical data from patients with colorectal cancer in various stages of the disease. A diagnostic test was developed that allows selection of CMS4 tumors for therapeutic purposes. With this test, multiple regions within a single cancer were tested, and it was found that one tumor can contain multiple molecular subtypes. Furthermore, primary cancers and metastases can differ in molecular subtype, and chemotherapeutic treatment can influence the subtype. Finally, with the use of organoids, two promising novel treatments against poor-prognosis colorectal cancer were discovered. The tyrosine kinase inhibitor dasatinib attenuates invasion of tumor cells driven by the microenvironment and could thus improve treatment of CMS4 cancers. Inhibition of the protein ATR increases the efficacy of intraperitoneal chemotherapy treatment by preventing DNA damage repair in tumor cells. Molecular classification of colorectal cancer can be implemented in clinical practise, and can contribute to the development of novel (subtype-targeted) therapeutic strategies against poor-prognosis colorectal cancer

A potential role for CCN2/CTGF in aggressive colorectal cancer

CCN2, also known as connective tissue growth factor (CTGF) is a transcriptional target of TGF-β signaling. Unlike its original name ("CTGF") suggested, CCN2 is not an actual growth factor but a matricellular protein that plays an important role in fibrosis, inflammation and connective tissue remodeling in a variety of diseases, including cancer. In pancreatic ductal adenocarcinoma, CCN2 signaling induces stromal infiltration and facilitates a strong tumor-stromal interaction. In many types of cancer, CCN2 overexpression has been associated with poor outcome. CMS4 (Consensus Molecular Subtype 4) is a recently identified aggressive colorectal cancer subtype, that is characterized by up-regulation of genes involved in epithelial-to-mesenchymal transition, TGF-β signaling, angiogenesis, complement activation, and extracellular matrix remodeling. In addition, a high influx of stromal fibroblasts contributes to the mesenchymal-like gene expression profile of this subtype. Furthermore, compared with the other three CMS groups, CMS4 tumors have the worst prognosis. Based on these observations, we postulated that CCN2 might contribute to colorectal cancer progression, especially in the CMS4 subtype. This review discusses the available literature on the role of CCN2 in colorectal cancer, with a focus on the 'fibrotic subtype' CMS4

In Patients Undergoing CRS/HIPEC for Colorectal Adenocarcinoma with Peritoneal Metastases, Presence of Ascites on Computed Tomography Imaging is not a Prognostic Marker for Survival

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially curative treatment for patients with colorectal peritoneal metastases (CRPM). Patient selection is key to optimizing outcomes after CRS/HIPEC. The aim of this study was to determine the prognostic value of ascites diagnosed on preoperative imaging. METHODS: A prospective database of patients eligible for CRS/HIPEC between 2010 and 2020 was retrospectively analyzed. The presence of ascites, postoperative complications, overall survival (OS), disease-free survival (DFS), and completeness of cytoreduction were assessed. Univariable and multivariable logistic regression was performed to identify independent predictors for outcome. RESULTS: Of the 235 included patients, 177 (75%) underwent CRS/HIPEC while 58 (25%) were not eligible for CRS/HIPEC. In 42 of the 177 patients (24%) who underwent CRS/HIPEC, ascites was present on preoperative computed tomography (CT) imaging. Peritoneal Cancer Index (PCI) score was significantly higher in patients with preoperative ascites compared with patients without (11 [range 2-30] vs. 9 [range 0-28], respectively; p = 0.011) and complete cytoreduction was more often achieved in patients without ascites (96.3% vs. 85.7%; p = 0.007). There was no significant difference in median DFS and OS after CRS/HIPEC between patients with and without ascites {10 months (95% confidence interval [CI] 7.1-12.9) vs. 9 months (95% CI 7.2-10.8), and 25 months (95% 9.4-40.6) vs. 27 months (95% CI 22.4-31.6), respectively}. CONCLUSIONS: Ascites on preoperative imaging was not associated with worse survival in CRS/HIPEC patients with CRPM. Therefore, excluding patients from CRS/HIPEC based merely on the presence of ascites is not advisable

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFβ signaling in colorectal cancer

Abstract Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFβ1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFβ signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFβ signaling, while KIT deletion sensitized PDOs to TGFβ-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFβ signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFβ expression, and protects the tumor cells from tumor-suppressive TGFβ signaling by inhibiting SMAD2 expression

Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells

Background & Aims Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. Methods We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. Results Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT+ colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. Conclusions Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia

Tumor Seeding During Colonoscopy as a Possible Cause for Metachronous Colorectal Cancer

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A Novel Diagnostic Tool for Selecting Patients With Mesenchymal-Type Colon Cancer Reveals Intratumor Subtype Heterogeneity

Background: Consensus molecular subtype 4 (CMS4) is a recently identified aggressive colon cancer subtype for which platelet-derived growth factor receptors (PDGFRs) and KIT are potential therapeutic targets. We aimed to develop a clinically applicable CMS4 reverse transcription polymerase chain reaction (RT-qPCR) test to select patients for PDGFR/KIT-targeted therapy. Methods: We used logistic regression to develop a CMS4 prediction rule based on microarray expression values of PDGFRA , PDGFRB , PDGFC , and KIT (566 training and 1259 test samples, using the 273-gene random forest classifier as CMS4 reference standard). We next translated the prediction rule into a single-sample RT-qPCR test, which we independently validated in 29 fresh tumor samples. To study intratumor CMS4 heterogeneity, we used the RT-qPCR test to analyze five random regions of 20 colon tumors. Results: The microarray-based prediction rule diagnosed CMS4-type tumors extremely well in both training and independent test samples (training: area under the curve [AUC] = 0.95, 95% confidence interval [CI] = 0.94 to 0.97; test: AUC = 0.95, 95% CI = 0.94 to 0.96), with excellent calibration and approximately 80% overall net benefit over a large threshold range. Translation into an RT-qPCR test did not affect discrimination (AUC = 0.97, 95% CI = 0.93 to 1.00, independent validation). RT-qPCR analysis of five random tumor regions revealed extensive intratumor CMS4 heterogeneity in nine out of 20 tumors. At least two regions likely have to be analyzed to identify patients that are predominantly CMS4 positive (>50% average CMS4 chance). Conclusion: The CMS4 RT-qPCR test is a promising clinical tool for selecting individual patients for CMS4-subtype-targeted therapy