A LCK/PPARG/SCD Lipid Metabolism Axis Promotes Invasion in Human Oral Cancer

In previous studies, we leveraged intratumoral heterogeneity of oral cancer cells to isolate clonal lines with distinct invasive properties. Integrative expression analyses identified lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver and therapeutic target for metastasis. However, its downstream mechanisms remained unclear.

Here, we show that LCK modulates expression of peroxisome proliferator-activated receptor gamma (PPARG), a key regulator of lipid metabolism. Using 3D invasion assays, untargeted lipidomics, and transcriptomics, we found that PPARG activity reduced lipid storage while enhancing fatty acid synthesis, membrane remodeling, and cellular invasiveness downstream of LCK. Notably, PPARG upregulated stearoyl-CoA desaturase (SCD), which converts saturated to monounsaturated fatty acids, thereby limiting lipid peroxide accumulation, a trigger of ferroptosis.

Importantly, SCD inhibition reduced cancer cell viability, particularly upon PPARG activation by rosiglitazone. Together, our findings identify an previously undescribed oncogenic LCK/PPARG/SCD axis that modulates lipid composition to promote metastasis. While this gives concerning context to ongoing studies of PPARG activation as anti-carcinogenic, targeting this signaling pathway offers a promising strategy for anti-metastatic therapy in oral cancer.

Sample collection and extraction for untargeted lipidomics analysis 4x10^5 cells were grown for 24 h on a 6-well plate were gently scraped off the plates in PBS, centrifuged and resuspended in 150 µl water. Consequently, 200 µl methanol, 400 µl chloroform and 15 µl 1 M hydrochloric acid were added and the solution was vortexed and incubated on a shaker for 1 h at 550 rpm. Subsequently, after centrifugation at 2000 g for 10 min, the organic phase was transferred to a new vial.

The aqueous phase was mixed with another 100 µl methanol, 200 µl chloroform and 10 µl 1 M hydrochloric acid and extraction was repeated. The combined organic phases were dried under a nitrogen stream and dissolved in 50 µL dichloromethane. LC-MS/MS analysis The employed frontend was a Thermo Fisher Scientific Vanquish H UHPLC with a Waters Acquity BEH C18 column (150 mm × 1 mm × 1.7 µm; Waters Corporation, Milford, MA, USA).

Mobile phase A was 60% acetonitrile in aqueous 10 mM ammonium formate; mobile phase B was 10% acetonitrile in isopropanol supplemented with 10 mM ammonium formate. The following gradient was employed at a constant flow rate of 70 µL/min and 40 °C: 0 min; 2% B, 7 min; 2% B, 20 min; 100% B, 25 min; 100% B, followed by 5 min re-equilibration to starting conditions. Lipids were analyzed employing an untargeted lipidomics workflow with separate positive and negative polarity runs on a Bruker timsTOF Pro equipped with a VIP-HESI source (Bruker Corporation, Billerica, MA, USA).

Mass spectrometry (MS) was operated in PASEF mode (collision energy 30 eV) with a full scan in the range of 100-2200 m/z followed by two PASEF ramps resulting in a total cycle time of 0.32 s. Data Processing Data were analyzed with Bruker Compass MetaboScape 2022b Version 9.0.1 (Build 11878). Features were annotated by the rule based MCube LipidSpecies annotation algorithm and the MS-DIAL LipidBlast (version 68) spectral databases.