Jin Gu

Mucinous adenocarcinoma (MAC) represents a subtype of colorectal cancer (CRC) characterized by insensitivity to chemoradiotherapy, necessitating urgent development of novel therapeutic strategies specifically targeting tumor biology of MAC. Integrated analysis of ATAC-seq and RNA-seq data was performed to identify pivotal targets mediating treatment resistance in MAC. Subsequently, clinical specimens were collected for immunohistochemistry, RT-qPCR, and Kaplan-Meier survival analysis. Functional validation of the target was conducted through in vitro experiments encompassing colony formation, drug sensitivity assessments, synergy testing, and immunofluorescence. The translational potential of the target was evaluated in vivo. Integrated ATAC-seq and RNA-seq analyses identified hypoxia and dysregulated ferroptosis as critical features of MAC, screening carbonic anhydrase 9 (CA9) as a pivotal gene implicated in MAC treatment resistance. CA9-specific inhibitor synergized with 5-fluorouracil to exert enhanced antitumor effects. Additionally, CA9 knockdown or inhibition arrested tumor cell proliferation and migration, promoted intracellular reactive oxygen species generation, induced mitochondrial shrinkage, increased mitochondrial iron content, reduced glutathione levels, and triggered lipid peroxidation. Inhibitors of either ferroptosis or apoptosis antagonized CA9 inhibitor-mediated cell death. In vivo experiments demonstrated that CA9 knockdown or inhibition significantly delayed tumor growth. Co-immunoprecipitation revealed that CA9 interacts directly or indirectly with multiple ferroptosis-associated proteins. This study identifies the hypoxic tumor microenvironments and dysregulated ferroptosis as pivotal molecular characteristics of MAC, and proposes a novel mechanism underlying treatment resistance in MAC: Hypoxia remodels chromatin accessibility through epigenetic modifications, to dysregulate CA9 expression, which may subsequently modulate cellular susceptibility to ferroptosis, culminating in treatment resistance, and targeting CA9 may improve the therapeutic efficacy of MAC, although further studies are needed to establish direct causality.

Validation that AKT3 overexpression enhances the invasion and migration capabilities of colorectal cancer cells.

Colorectal ovarian metastasis (CROM), a distinct metastatic subtype of colorectal cancer, is associated with early disease onset and aggressive progression. CROM lacks specific treatment options, highlighting the need to elucidate the underlying biological mechanisms and potential therapeutic vulnerabilities. In this study, we performed integrated analyses of single-cell RNA sequencing (scRNA-seq) datasets from 155,163 cells across 35 patients from the in-house cohort and public datasets, with matched bulk transcriptomic profiling. The analysis identified AKT3+ epithelial–mesenchymal transition (EMT)-like cells at the invasive tumor–stroma interface as metastasis-initiating cells. Functional validation using in vivo xenograft models demonstrated that AKT3 deficiency reduced ovarian colonization, whereas AKT3 overexpression conferred a mesenchymal phenotype with invasive capacity. Furthermore, reciprocal cross-talk between AKT3+ mesenchymal-like cells and cancer-associated fibroblasts (CAF) played a key role in remodeling the tumor microenvironment. Multiplex immunofluorescence staining of primary tumor specimens revealed spatially coordinated AKT3+/SNAIL+/ITGB1+ tumor buds adjacent to αSMA+ CAFs at the invasive front. Critically, AKT3 inhibition or knockdown in patient-derived CROM organoids (CROM-PDO) significantly suppressed malignant phenotypes, recapitulating the AKT3 dependency. Collectively, these findings elucidate an AKT3-driven feedforward loop coupling EMT plasticity with CAF activation as a critical driver of CROM and propose CROM-PDOs as a robust platform for developing precision therapies targeting this aggressive colorectal cancer subtype.Significance:Single-cell analyses reveal AKT3-expressing mesenchymal-like cells as drivers of colorectal ovarian metastases, which depends on the dynamic cross-talk between cancer cells and cancer-associated fibroblasts within the immunosuppressive ovarian niche.

ScRNA-seq quality control metrics and cell type distribution in colorectal ovarian metastasis (CROM) samples.

The inhibitory effect of the AKT inhibitor Uprosertib on the proliferation and migration of CRC cell lines (HT29 and SW480).

Pathway enrichment analysis and validation of AKT3, SNAIL, and CDH2 expression in CROM patient samples and organoids.

In vivo assessment of ovarian metastasis rates and tumor burden in mice following AKT3 knockdown or overexpression.

The effect of Uprosertib treatment and AKT3 knockdown on the viability and growth of patient-derived organoids (PDOs).

The effect of AKT3 knockdown on the proliferation, migration, invasion, and cytoskeletal structure of CRC cell lines.