Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted ...agents. More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers). The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype-positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor-targeted therapy.
Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, ...which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including
ASCL2 and
LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.
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► Wnt target genes, including stem cell markers, inversely correlate with prognosis ► Progression of CRC is associated with methylation of Wnt target genes ► Adherence to CSC signatures reflects an immature clonal trait, not CSC numbers ► Wnt target methylation is a strong prognostic marker
Despite the presence of mutations in APC or beta-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when ...beta-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate beta-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo. We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.
Patient‐derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor ...heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.
What's new?
Patient‐derived xenograft (PDX) models have become an important asset in translational cancer research. However, colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features, and to what extent the existing CRC PDX collection represents the inter‐patient heterogeneity remains an open question. This study identifies a subtype‐specific bias in the establishment of PDXs from CRC patients, leaving the major subtype CMS2 strongly underrepresented. Additionally, the findings suggest that further classification within CMS can be achieved. For CMS2, the proliferation‐related marker Ki67 may thus help refine patient classification, estimate prognosis, and guide treatment decisions.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundance of stroma. Multiple molecular classification efforts have identified a mesenchymal tumor subtype that is consistently ...characterized by high‐grade growth and poor clinical outcome. The relation between PDAC stroma and tumor subtypes is still unclear. Here, we aimed to identify how PDAC cells instruct the main cellular component of stroma, the pancreatic stellate cells (PSCs). We found in primary tissue that high‐grade PDAC had reduced collagen deposition compared to low‐grade PDAC. Xenografts and organotypic co‐cultures established from mesenchymal‐like PDAC cells featured reduced collagen and activated PSC content. Medium transfer experiments using a large set of PDAC cell lines revealed that mesenchymal‐like PDAC cells consistently downregulated ACTA2 and COL1A1 expression in PSCs and reduced proliferation. We identified colony‐stimulating factor 1 as the mesenchymal PDAC‐derived ligand that deactivates PSCs, and inhibition of its receptor CSF1R was able to counteract this effect. In conclusion, high‐grade PDAC features stroma that is low in collagen and activated PSC content, and targeting CSF1R offers direct options to maintain a tumor‐restricting microenvironment.
Synopsis
This study reveals that PDAC cells counteract mechanical restrictions of tumor stroma by secreting CSF‐1, which could be a potential therapeutic target.
Poorly differentiated, high‐grade pancreatic cancers, corresponding with the mesenchymal subtype, feature stroma that is low in collagen and activated pancreatic stellate cell content.
These mesenchymal subtype cancer cells secrete high amounts of CSF‐1.
CSF‐1 downregulates stromal activation markers and reduces proliferation via STAT3 in PSCs.
Depletion of CSF‐1 in high‐grade PDAC abolishes PSC deactivation.
High‐grade CSF‐1‐positive PDAC patient samples exhibit deactivated stroma.
This study reveals that PDAC cells counteract mechanical restrictions of tumor stroma by secreting CSF‐1, which could be a potential therapeutic target.
High expression of cancer stem cell (CSC) marker CD133 has been used as a predictor for prognosis in colorectal cancer (CRC), suggesting that enumeration of CSCs, using CD133, is predictive for ...disease progression. However, we showed recently that both CD133 mRNA and protein are not downregulated during differentiation of colon CSCs, pointing to an alternative reason for the prognostic value of CD133. We therefore set out to delineate the relation between CD133 expression and prognosis.
A CRC patient series was studied for expression of CD133 and other CSC markers by microarray and quantitative PCR analysis. In addition, several common mutations were analyzed to determine the relation with CD133 expression.
CD133 mRNA expression predicted relapse-free survival in our patient series, whereas several other CSC markers could not. Moreover, no correlation was found between expression of other CSC markers and CD133. Interestingly, high CD133 expression was related to mutations in K-Ras and B-Raf, and inhibition of mutant K-Ras or downstream mitogen-activated protein kinase kinase (MEK) signaling decreases CD133 expression. In addition, an activated K-Ras gene expression signature could predict CD133 expression in our patient set as well as data sets of other tumor types.
CD133 expression is upregulated in CRC tumors that have a hyperactivated Ras-Raf-MEK-ERK pathway and is therefore related to mutations in K-Ras or B-Raf. As mutations in either gene have been related to poor prognosis, we conclude that CD133 expression is not indicative for CSC numbers but rather related to the mutation or activity status of the Ras-Raf pathway.
Colorectal cancer (CRC) is a heterogeneous disease that can currently be subdivided into four distinct consensus molecular subtypes (CMS) based on gene expression profiling. The CMS4 subtype is ...marked by high expression of mesenchymal genes and is associated with a worse overall prognosis compared to other CMSs. Importantly, this subtype responds poorly to the standard therapies currently used to treat CRC. We set out to explore what regulatory signalling networks underlie the CMS4 phenotype of cancer cells, specifically, by analysing which kinases were more highly expressed in this subtype compared to others. We found AKT3 to be expressed in the cancer cell epithelium of CRC specimens, patient derived xenograft (PDX) models and in (primary) cell cultures representing CMS4. Importantly, chemical inhibition or knockout of this gene hampers outgrowth of this subtype, as AKT3 controls expression of the cell cycle regulator p27
. Furthermore, high
expression was associated with high expression of epithelial-mesenchymal transition (EMT) genes, and this observation could be expanded to cell lines representing other carcinoma types. More importantly, this association allowed for the identification of CRC patients with a high propensity to metastasise and an associated poor prognosis. High
expression in the tumour epithelial compartment may thus be used as a surrogate marker for EMT and may allow for a selection of CRC patients that could benefit from AKT3-targeted therapy.
Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal ...infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020).
To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns.
PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2.
Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.
DNA hypermethylation is common in colon cancer. Previously, we have shown that methylation of WNT target genes predicts poor prognosis in stage II colon cancer. The primary objective of this study ...was to assess whether pre-operative treatment with decitabine can decrease methylation and increase the expression of WNT target genes APCDD1, AXIN2 and DKK1 in colon cancer patients. A clinical study was conducted, investigating these potential effects of decitabine in colon cancer patients (DECO). Patients were treated two times with 25 mg/m2 decitabine before surgery. Methylation and expression of LINE1 and WNT target genes (primary outcome) and expression of endogenous retroviral genes (secondary outcome) were analysed in pre- and post-treatment tumour samples using pyrosequencing and rt-PCR. Ten patients were treated with decitabine and eighteen patients were used as controls. Decitabine treatment only marginally decreased LINE1 methylation. More importantly, no differences in methylation or expression of WNT target or endogenous retroviral genes were observed. Due to the lack of an effect on primary and secondary outcomes, the study was prematurely closed. In conclusion, pre-operative treatment with decitabine is safe, but with the current dosing, the primary objective, increased WNT target gene expression, cannot be achieved.
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