A Study on the Functional Role of Hsp90α in Head and Neck Cancer Invasion
Background: Many cellular processes are involved in maintaining the cancer microenvironment. Hsp90α is up-regulated in a number of different cancer tissues including breast cancer, prostate cancer, small cell lung cancer, acute myeloid leukemia among others, and it has been regarded as a potent cancer biomarker through stabilizing the oncogenic proteins integrity and functionally. Previous studies showed that Hsp90α inhibition reduces many key oncogenic proteins responsible for cancer signaling, proliferation, invasion and survival. Therefore, identifying a potential role of Hsp90 in the head and neck squamous cell carcinoma phenotype followed with identifying Hsp90α client proteins is a rational approach to further understand the underlying process of head and neck squamous cell carcinoma (HNSCC). A preliminary study conducted in our lab showed the transcription factor SOX11 to be up-regulated in the highly invasive HNSCC cancer cell lines versus low invasive ones, through which we hypothesized that SOX11 might be a potential new client protein for Hsp90α.Objective: To study the functional role of Hsp90α in the invasiveness, motility and survival of the HNSCC cells and to discover a new client protein to further understand the mechanism and mode of action of the Hsp90α in HNSCC cell lines.
Aim #1: To analyze and compare the expression of the cytoplasmic and membranous Hsp90α among different HNSCC cell lines.
Aim #2: To identify the functional role of Hsp90α on the phenotypic traits of the invasive HNSCC cells.
Aim #3: To identify the functional biochemical interaction between Hsp90α as a chaperone and SOX11 as a potential client protein.
Aim #4: To determine a potential SOX11 up-stream regulatory role on Hsp90α.
Materials and Methods: Western Blotting and qPCR were used to assess the expression of Hsp90α among four HNSCC cell lines, UM1, UM2, UM5 and UM6. Phenotypic studies including proliferation, migration and invasion assays were performed after a siRNA knockdown of Hsp90α to investigate the functional role of Hsp90α in HNSCC cells. We performed a Co-IP assay using SOX11 antibody to pull down SOX11 and its associated proteins on a proteomic scale and use liquid chromatography mass spectrometry (LC-MS/MS) to identify the binding proteins that have possible functional interaction with SOX11. A Co-IP experiment using SOX11 antibody followed by Western blot analysis of Hsp90α was used to confirm the MS results, and a second Co-IP experiment using Hsp90α antibody followed with Western blot analysis of SOX11 was conducted to further confirm the binding between Hsp90α and SOX11. siRNA knock down of SOX11 was performed to assess if down-regulation of SOX11 affects the expression of Hsp90α in HNSCC cells.
Result: Both Western blotting and qPCR indicated that Hsp90α is over-expressed in the invasive UM1 and UM5 cell lines when compared to low invasive UM2 and UM6 cell lines (p<0.05). Hsp90α transient knock down in highly invasive UM1 and UM5 cell lines to assess the role of Hsp90α on proliferation, migration and invasion resulted in a proliferation assay showing a declined proliferation course in the knockdown set (p < 0.05). Wound healing assay showed a 40% decrease in UM1 migration potency (p < 0.001). Similarly, invasion assay showed 99% (UM1) and 89.2% (UM5) decrease in invasion potential (p < 0.001). LC-MS/MS analysis of the Co-IP samples of UM1 and UM5 cell lines showed that Hsp90α is among the high abundant proteins that bind to SOX11. The MS data was confirmed by a Co-IP assay using SOX11 antibody followed by Western blot analysis of Hsp90α, which shows the potential binding between SOX11 and Hsp90α. The second Co-IP assay conducted using Hsp90α antibody and Western blot analysis of SOX11 also confirmed the binding between the two proteins. Finally, After the knockdown of SOX11 in UM1 and UM5 cells with siRNA, there was no significant reduction of Hsp90α levels.
Conclusion: Hsp90α may play an important functional role in the invasiveness of HNSCC cells. SOX11 could be a potential client protein for Hsp90α. the transcriptional factor SOX11 may not directly regulate the expression of Hsp90.