Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

AKT3 Expression in Mesenchymal Colorectal Cancer Cells Drives Growth and Is Associated with Epithelial-Mesenchymal Transition.

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 p27KIP1. Furthermore, high AKT3 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 AKT3 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.

Exploring and modelling colon cancer inter-tumour heterogeneity: opportunities and challenges.

Colon cancer inter-tumour heterogeneity is installed on multiple levels, ranging from (epi)genetic driver events to signalling pathway rewiring reflected by differential gene expression patterns. Although the existence of heterogeneity in colon cancer has been recognised for a longer period of time, it is sparingly incorporated as a determining factor in current clinical practice. Here we describe how unsupervised gene expression-based classification efforts, amongst which the consensus molecular subtypes (CMS), can stratify patients in biological subgroups associated with distinct disease outcome and responses to therapy. We will discuss what is needed to extend these subtyping efforts to the clinic and we will argue that preclinical models recapitulate CMS subtypes and can be of vital use to increase our understanding of treatment response and resistance and to discover novel targets for therapy.

Consensus molecular subtypes of colorectal cancer are recapitulated in in vitro and in vivo models.

Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.

Protein kinase C α is a central signaling node and therapeutic target for breast cancer stem cells.

The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs.

Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients.

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.