Exploration of the role of FOSL1-mediated liquid-liquid phase separation in cisplatin resistance in HNSCC

About This Grant

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, with over 890,000 new cases annually. However, there have been few advancements in treatments for HNSCC over the past decade, leaving cisplatin to remain the standard chemotherapeutic agent. Despite the efficacy and affordability of cisplatin, resistance is a major setback in the successful treatment of HNSCC. Many HNSCC patients experience relapse and develop a resistance to cisplatin, diminishing its effectiveness over time. Cisplatin resistance (CR) can occur through various mechanisms, including the enrichment of cancer stem cells (CSCs). This process could be facilitated through liquid-liquid phase separation (LLPS), resulting in CR. In cells, LLPS regulates various biochemical processes by forming membrane-less condensates. This process is controlled by intrinsically disordered regions (IDRs), in which their flexibility allows them to interact with multiple binding partners to control various cellular functions. Recent studies have linked LLPS to the formation of super- enhancers (SEs), in which this interplay is crucial for cell identity and tumorigenesis. FOSL1 (Fos-like Antigen- 1) is a protein that plays a significant role in regulating gene expression, cell proliferation, and differentiation. Structurally, FOSL1 has two IDRs that could contribute to LLPS with other regulators and transcription factors. In our previous studies, we have shown that FOSL1 is upregulated in HNSCC CSCs and revealed that FOSL1 establishes SEs to maintain the high expression of cancer stemness genes (e.g., SNAI2, CD44, and FOSL1 itself) to promote tumorigenesis and metastasis. Additionally, we found that FOSL1 promotes CR by enhancing the CSC population in HNSCC mouse models. Furthermore, we demonstrated that FOSL1 can undergo phase separation with other key regulators to establish SEs. In our preliminary studies, we observed an increase in FOSL1 condensate formation in CSCs compared to non-CSCs in HNSCC. We also demonstrated that IDRs in FOSL1 are required for its phase separation capabilities. Based on these findings, we hypothesize that FOSL1 undergoes LLPS at SEs associated with cancer stemness genes to promote CSC maintenance and facilitate CR in HNSCC. To test this hypothesis, the following aims will be investigated: (1) Aim 1 will identify key regulators involved in FOSL-dependent LLPS in HNSCC. This will be achieved using FOSL1 recombinant proteins and nuclear extract of HNSCC CSCs to obtain FOSL1 nuclear condensates and identify the key regulators that partition with FOSL1 LLPS through proteomic analysis. ChIP-seq and RNA-seq will be used to identify key cancer stemness genes in HNSCC CSCs. Moreover, immunofluorescence and RNAscope will be used to determine if the regulators and oncogenes co-localize within the FOSL condensate; (2) Aim 2 will examine the functionality of the FOSL1 mutant without LLPS ability and its capabilities of promoting tumorigenesis, metastasis, and CR in vitro and in vivo. These findings will elucidate how FOSL1-dependent LLPS maintains the functional properties of CSCs to promote CR in HNSCC and identify potential drug targets to improve the efficacy of chemotherapies.