ALPK2 promotes cell growth via the AP-1 axis in YAP/TAZ-activated oral squamous cell carcinoma

Technical Summary: ALPK2 Promotes Cell Growth via the AP-1 Axis in YAP/TAZ-Activated Oral Squamous Cell Carcinoma

Problem Statement
Oral squamous cell carcinoma (OSCC) remains a significant clinical challenge, with advanced or metastatic cases carrying a poor prognosis. The Hippo signaling pathway, specifically the aberrant activation of its effectors YAP and TAZ, is a known driver of OSCC proliferation and progression. While the upstream mechanisms activating YAP/TAZ (such as EGFR amplification or FAT1 loss) are partially understood, the full spectrum of downstream transcriptional programs regulated by YAP/TAZ in OSCC remains largely unclear. Furthermore, direct therapeutic targeting of YAP/TAZ is difficult due to their nature as transcriptional co-activators lacking catalytic sites, and upstream kinases (MST1/2, LATS1/2) function as tumor suppressors, making their inhibition counterproductive. Consequently, there is an urgent need to identify novel, druggable downstream mediators of YAP/TAZ activation that could serve as therapeutic targets.

Methodology
The study employed a multi-faceted approach to identify and validate YAP/TAZ-regulated proliferation genes and their downstream signaling mechanisms:

• Genomic Screening: RNA sequencing (RNA-seq) was performed on LATS1/2 knockout (KO) CAL27 cells (which exhibit constitutive YAP/TAZ activation) compared to wild-type (WT) controls to identify upregulated kinases.
• Bioinformatic Analysis: Gene Set Enrichment Analysis (GSEA) was conducted using data from the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) to correlate ALPK2 expression with YAP/TAZ activity signatures across cancer cell lines and tissues.
• Functional Validation: Cell proliferation was assessed via crystal violet staining and CellTiter-Blue viability assays in OSCC cell lines (WSU-HN6, CAL27) and EGFR-mutant lung adenocarcinoma lines (HCC827, PC-9) following ALPK2 knockdown (KD) via siRNA or shRNA.
• Mechanistic Elucidation:
  • Phosphoproteomics: Label-free quantitative phosphoproteomic analysis (LC-MS/MS) was used to identify changes in protein phosphorylation upon ALPK2 KD.
  • Transcriptomics: RNA-seq and GSEA were utilized to map downstream gene expression changes, specifically focusing on AP-1 family members.
  • Western Blotting: Protein levels and phosphorylation states of key signaling molecules (ALPK2, MTA2, YB-1, FOSL1, ERK) were verified across multiple cell lines.
  • Pharmacological Inhibition: Cells were treated with trametinib (a MEK1/2 inhibitor) to assess the sensitivity of YAP/TAZ-activated cells and the role of the MAPK/ERK pathway.

Key Contributions and Results

1. Identification of ALPK2 as a YAP/TAZ Target: RNA-seq analysis of LATS1/2 KO cells revealed ALPK2 (alpha-protein kinase 2) as the most significantly upregulated kinase. GSEA confirmed that high ALPK2 expression is strongly enriched in YAP/TAZ-activated gene signatures across both OSCC and lung adenocarcinoma datasets.
2. ALPK2 Drives Proliferation: Knockdown of ALPK2 significantly inhibited cell proliferation in OSCC cell lines (WSU-HN6, CAL27) and EGFR-mutant lung adenocarcinoma cells (PC-9, HCC827) with activated YAP/TAZ. This effect was observed in both transient siRNA and stable shRNA models.
3. Dissection of the Downstream Pathway:
   • Exclusion of PI3K/AKT: Unlike previous reports in ovarian and renal cancers, ALPK2 KD in OSCC did not alter AKT or S6 phosphorylation, suggesting a distinct downstream mechanism in this context.
   • MTA2 and AP-1 Axis: Phosphoproteomics identified MTA2 (a component of the NuRD complex) as a protein with significantly reduced phosphorylation upon ALPK2 KD. Subsequent RNA-seq and GSEA indicated that ALPK2 KD led to the downregulation of AP-1 family genes, specifically FOSL1 (FRA-1).
   • FOSL1 as a Critical Effector: FOSL1 expression was consistently reduced by ALPK2 KD across all tested cell lines. Functional assays confirmed that FOSL1 KD alone significantly suppressed cell growth.
   • MAPK Connection: Treatment with trametinib (MEK inhibitor) reduced FOSL1 levels, and LATS1/2 KO cells (high YAP/TAZ) showed increased sensitivity to trametinib, linking the ALPK2-MTA2 axis to MAPK/ERK signaling.
4. Proposed Mechanism: The authors propose a regulatory axis where YAP/TAZ activation upregulates ALPK2 transcription. ALPK2 then influences the phosphorylation status of MTA2, which in turn modulates AP-1 transcriptional activity (specifically FOSL1), driving tumor proliferation.

Significance and Claims
The study claims to be the first to demonstrate the involvement of ALPK2 in OSCC progression via the YAP/TAZ pathway and to suggest a link with the MTA2-AP-1 signaling axis. The authors posit that ALPK2 represents a novel therapeutic vulnerability in YAP/TAZ-active cancers.

• Therapeutic Potential: Since YAP/TAZ themselves are difficult to target directly, and upstream Hippo components are tumor suppressors, ALPK2 offers a promising intermediate target. As a non-typical kinase with a unique alpha-kinase domain, ALPK2 may be amenable to small-molecule inhibitor development.
• Clinical Relevance: The findings suggest that ALPK2 could serve as a biomarker for YAP/TAZ activation and a target for precision medicine, potentially in combination with other molecularly targeted therapies (e.g., EGFR inhibitors).
• Caveats and Limitations: The authors modestly note that the direct causal relationship between ALPK2, MTA2, and YB-1 requires further mechanistic verification (e.g., via co-immunoprecipitation or kinase assays) to confirm direct phosphorylation. Additionally, they acknowledge that systemic inhibition of ALPK2 carries a theoretical risk of cardiac side effects (specifically impaired diastolic function), necessitating tumor-specific delivery strategies for future therapeutic development. The study also highlights that whether YAP/TAZ directly bind the ALPK2 promoter remains to be confirmed via chromatin immunoprecipitation.