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E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

ABSTRACT: The epithelial-mesenchymal transition (EMT) is closely linked to the acquisition of cancer stem cell (CSC) properties, which contribute to treatment resistance and metastasis. This study investigates the role of the E3 ubiquitin-ligase Hakai, the first identified post-translational regulator of E-cadherin stability, in promoting CSC traits in colorectal cancer (CRC). To examine Hakai’s involvement in CSC regulation, we used an inducible shRNA in a HT29 cells. Under conditions that promote CSC characteristics, we silenced Hakai and evaluated tumoursphere formation and CSC marker expression. Proteomic and bioinformatic analyses were performed to identify Hakai-regulated proteins in tumoursphere cultures. Additionally, Western blot, RT-qPCR, co-immunoprecipitation, immunofluorescence and TOPFlash assays were employed to study CSC-related protein regulation in response to Hakai expression. Furthermore, we assessed the impact of Hakin-1, the pharmacological inhibitor specifically targeting Hakai’s HYB domain responsible for its E3 ubiquitin-ligase activity, on tumoursphere properties. Hakai silencing significantly reduced tumoursphere size and number accompanied by decreased expression of CSC markers and Wnt target genes. CSC-related proteins regulated by Hakai were identified, including LRP4, a negative regulator of Wnt/β-catenin pathway. Hakai interacts with LRP4, promoting its ubiquitination and degradation. Moreover, Hakai overexpression induces hyperactivation of Wnt/β-catenin sand disrupts LRP4’s inhibitory effect. Treatment with Hakin-1 effectively inhibited self-renewal and promoted differentiation within tumourspheres. These findings suggest that Hakai promotes CSCs properties by hyperactivation of the Wnt/β-catenin pathway via LRP4-mediated modulation. Additionally, Hakin-1 emerges as a promising therapeutic agent targeting CSCs by enhancing differentiation and attenuating Wnt/β-catenin activity, highlighting Hakai as a potential target for improving CSC treatment.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: María Pereira Blanco

LAB HEAD: Angélica Figueroa

PROVIDER: PXD069248 | Pride | 2026-06-22

REPOSITORIES: Pride

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			Action	DRS
	Tumorspheres_shCbll1_N1.d.zip	Other
	Tumorspheres_shCbll1_N2.d.zip	Other
	Tumorspheres_shCbll1_N3.d.zip	Other
	Tumorspheres_shControl_N1.d.zip	Other
	Tumorspheres_shControl_N2.d.zip	Other

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Project description:Increasing evidence suggests that cancer arises from cells that are capable of initiating and sustaining neoplastic tissue growth, termed cancer stem cells (CSCs). Of central scientific and clinical relevance, cells with CSC properties are enriched for chemo- and radiation resistance and therefore may represent a population of cells that must be therapeutically targeted to prevent cancer recurrence/relapse 1. Human CSCs were first isolated in neoplastic hematopoietic tissue that manifests leukemias such as adult acute myeloid leukemia (AML) 2. AML stem cells represent a benchmark model of human CSC biology, ultimately motivating foundational studies leading to the identification of CSCs from solid tumours such as breast and colon 3. Independent of tissue type, a consistent feature of CSCs is their uncontrolled self-renewal capacity and differentiation blockade that have been commonly related to aberrant activation of pro-oncogenic events such as dysregulation of CBP/p300 transcriptional regulation involving β-catenin 4. However, the transcriptional networks involving CBP/p300/β-catenin complex have been shown to be equally critical to maintain normal stem cell (SCs) self-renewal for tissue homeostasis and regeneration 5. Here, we identify Sam68 as a distinct target that affords the ability to uniquely regulate CBP mediated transcription in human CSCs. Using a small molecule that targets Sam68, we reveal that shifting its affinity for CBP disrupts CBP/β-catenin complexes, leading to immediate changes in histone H3 (K14 and K18) acetylation. Chemical targeting of Sam68 induced global changes in transcriptional programs of patient AML cells involving apoptosis and differentiation and was able to uniquely reduce neoplastic self-renewal of human CSCs in an in vivo model of patient specific acute myeloid leukemia (AML). Our study establishes an approach whereby the CBP/β-catenin transcriptome can be uniquely targeted via Sam68 based vulnerability of CSCs that impacts neoplastic differentiation and self-renewal.

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E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

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