Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Survivin, a homo-dimeric protein, is a member of the inhibitor of apoptosis protein family. It is overexpressed in a wide spectrum of cancers, contributing to oncogenic evasion of apoptosis. USP1, a deubiquitinating enzyme, is often co-expressed with survivin in cancer cells. Ubiquitination and proteasomal degradation are critical for regulating CDK4/6 signaling. Here, we examine the relationship between CDK4/6 signaling, USP1 activation, and survivin upregulation in head and neck squamous cell carcinoma (HNSCC).
Methods: Two oral cavity HNSCC cell lines (CAL27 and HN5) and one conditionally reprogrammed cell culture of an oral cavity cancer (CR15) were used for the study experiments. RNAseq analysis and Western blots were used to examine mRNA and protein expression levels of survivin, along with a panel of BCL2 family signaling molecules related to apoptosis, after treatment with the CDK4/6 inhibitor palbociclib. The survivin dimerization inhibitor LQZ-7i, the proteasome inhibitor MG132, and ubiquitin specific peptidase 1 (USP1) inhibitor ML323 were also used to study the mechanism of survivin regulation. The protein-protein interactions were identified by co-immunoprecipitation. Protein subcellular localization was examined by immunofluorescence staining. Cell viability was measured by MTT assay. The reduction in target mRNA levels and protein levels was induced by siRNA knockdown.
Results: Survivin is consistently overexpressed in oral cavity squamous cell carcinoma cells and is predominantly localized in the nucleus. Survivin showed a cytoplasmic staining pattern after cells were treated with LQZ-7i. LQZ-7i also induced proteasome-dependent degradation of survivin, and MG132 treatment partially rescued the degradation. Furthermore, LQZ-7i inhibited cancer cell survival, with IC50 of 5.4 ± 1.7 µM in CAL27 and 4.2 ± 1.4 µM in HN5. Survivin was co-precipitated with USP1, which was also found in the nucleus, suggesting that survivin is a substrate of USP1. Knocking down the USP1 gene using siRNA led to a decrease in survivin protein levels; inhibition of USP1 with 40 µM ML323 selectively reduced survivin protein levels to ~5% in CAL27 and ~1% in CR15 after 3-day treatment. These data suggest that reduction in USP1 activity leads to an increase in ubiquitinated survivin population, thereby an increase in proteasomal degradation. ML323 also inhibited cancer cell survival, with IC50 of 26.7 ± 4.8 µM in CAL27 and 27.2 ± 0.8 µM in HN5. Furthermore, the inhibition of CDK4/6 by palbociclib selectively reduced mRNA levels of survivin and USP1, leading to a decrease in their protein levels. Palbociclib also induced a reduction in cancer cell survival, with IC50 of 2.8 ± 0.9 µM in CAL27 and 2.8 ± 0.1 µM in HN5.
Conclusion: Our work suggests that CDK4/6 upregulation (a common driving oncogenic event in HNSCC) may result in selective survivin upregulation via activation of USP1. This novel finding demonstrates that CDK4/6 signaling in HNSCC impacts oncogenic pathways beyond canonical proliferation signals.